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demonstrated effective elimination of primary tumors and disseminated metastases of FBL-3 tumors in B6 mice by a single iv injection of immune lymphocytes. These lymphocytes were resensitized in vitro to the FBL-3 tumor and expanded in Interleukin-2.
To make our approach more applicable to the clinical : situation in cancer patients, we investigated the efficacy of
adoptive transfer of fetal antigen-sensitized spleen cells on preestablished metastases. The primary tumor was allowed to grow for several days. After it had metastasized to the lungs, it was amputated; then the spleen cell transfers were started. Adoptive transfer of fetal antigen-sensitized spleen cells significantly inhibited metastases in mice in which the primary tumors were amputated. However, adoptive transfer of fetal antigen-sensitized spleen cells did not inhibit tumor growth or metastases in mice in which primary tumors were left intact. Another observation made was that the metastatic index in amputated control animals was significantly higher than that in the unamputated control group. It could be anticipated that surgery alone or adoptive transfer of immune cells alone is not beneficial in such tumors as this one. Surgery and repeated inoculation of fetal immune cells seem to improve antimetastatic therapy to a significant level in this system.
These findings on the role of oncofetal antigens in inhibiting tumor growth and metastases of a weakly immunogenic, metastatic tumor have direct relevance to human cancer, because the most commonly associated features of human cancers are their weak immunogenicity, their spontaneous metastatic behavior, and the need for therapy after the tumor has progressed beyond a certain stage.
[13] Cantor H, Boyse EA. Functional subclasses of T lymphocytes
bearing different Ly antigens. I. The generation of functionally distinct T cell subclass is a differentiative process independent of
antigen. J Exp Med 1975; 141:1376-1389. [14] Kisielow P, Hirst JA, Shiku H, et al. Ly antigens as markers for
functionally distinct subpopulations of thymus-derived lympho
cytes of the mouse. Nature 1975; 253:219-220. [15] JADINSKI J, Cantor H, TADAKUMA T, Peavy DL, Pierce CW. Sepa
ration of helper T cells from suppressor T cells expressing different Ly components. I. Polyclonal activation: Suppressor and helper activities are inherent properties of distinct T cell subsets. J Exp
Med 1976; 143:1382-1390. [16] HUBER B, DEVINSKY O, GERSHON RK, CANTOR H. Cell mediated
immunity: Delayed type hypersensitivity and cytotoxic responses are mediated by different T cell subclasses. J Exp Med 1976;
143:1534-1539. [17] VADAS MA, MILLER JF, MCKENZIE IF, et al. Ly and la antigen
phenotypes of T cells involved in delayed type hypersensitivity and
in suppression. J Exp Med 1976; 144:10-19. [18] BEVERLEY PC, Woody J, DUNKLEY M, FELDMAN M, McKenzie I.
Separation of suppressor and killer T cells by surface phenotypes.
Nature 1976; 262:495-497. [19] SHEN FW, McDougaL JS, BARD J, CORT SP. Developmental and
communicative interrelations of Ly123 and Lyl cell sets. J Exp
Med 1980; 151:566-572. [20] GAUTAM SC, Fyfe D, DEODHAR SD. Augmentation of immune re
sponse to syngeneic fibrosarcoma T241 with in vivo protection.
Cancer Immunol Immunother 1979; 6:81-88. [21] Crile G JR, DEODHAR SD. Role of pre-operative irradiation in
prolonging concomitant immunity and preventing metastases in
mice. Cancer 1971; 27:629-634. [22] Baldwin RW. Relevant animal models for tumor immunotherapy.
Cancer Immunol Immunother 1976; 1:197-198. [23] Martin DS, STOLFI R, FUGMAN RA. Animal models for tumor im
munotherapy. Cancer Immunol Immunother 1977; 2:77-79. [24] Howell SB, Dean JH, Esser EC, Law LN. Cell interactions in
adoptive immune rejection of a syngeneic tumor. Int J Cancer
1974; 14:662-674. [25] GAUTAM SC, Deodhar SD. Presence of suppressor cells in spleens of
mice bearing a weakly immunogenic syngeneic tumor. Cancer Res
1979; 39:2945-2951. [26] Fujimoto S, GREENE M, SEHON AH. Regulation of the immune
response to tumor antigens. I. Immuno-suppressor cells in tumor
bearing hosts. J Immunol 1976; 116:791-799. [27] BRODER S, WALDMANN TA. The suppressor-cell network in cancer. N
Engl J Med 1978; 299:1281-1284. [28] Fernandez-Cruz E, GILMAN SC, Feldman JD. Immunotherapy of a
chemically induced sarcoma in rats: Characterization of the effector T cell subset and nature of suppression. J Immunol 1982; 128:1112
1117. [29] BURTON RC, PLATE JM. Helper factor[s] augment in vitro induction
of tumor-specific immunity. Cell Immunol 1981; 58:225-237. [30] Shiku H, TAKAHASHI T, BEAN MA, OLD LJ, OETTGEN HF. Ly
phenotype of cytotoxic T cells for syngeneic tumor. J Exp Med
1976; 144:1116-1120. [31] STUTMAN O, SHEN FW, Boyse EA. Ly phenotype of T cells cytotoxic
for syngeneic mouse mammary tumors: Evidence for T cell inter
actions. Proc Natl Acad Sci USA 1977; 74:5667-5671. [32] Sinclair NR, McFarlane DL, Low JM. Cell mediated cytotoxicity
against syngeneic tumors. Cell Immunol 1981; 59:330-344. [33] Shimizu K, Shen FW. Role of different T cell sets in the rejection of
syngeneic chemically induced tumors. J Immunol 1979; 122:1162
1165. [34] Nelson M, Nelson DS, McKenzie IF, BLANDEN RV. Thy and Ly
markers on lymphocytes initiating tumor rejection. Cell Immunol
1981; 60:34-42. [35] BHAN AK, Perry LL, CANTOR H, McClusKEY RT, BENACERRAF B,
GREENE MI. The role of T cell sets in the rejection of a methylcholanthrene-induced sarcoma [S1509a] in syngeneic mice. Am J
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[1] Abelev GI. Production of embryonal serum alpha-globulin by hep
atomas. Review of experimental and clinical data. Cancer Res
1968; 28:1344-1350. [2] CINADER B. The future of tumor immunology. Med Clin N Am 1972;
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165. [4] Chism SE, BURTON RC, WARNER NL. Immunogenicity of oncofetal
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Page 3
not increase either tumor incidence or multiplicity in any resistant strain. Results similar to these were found by Witschi and Lock [5], although the effect of BHT on our BALB/cBy subline was much greater than the 50% increase they described for BALB/c mice.
We then sought to differentiate this genome-dependent effect on promotion from that on initiation by examining urethan dose-dependence in these same strains. When the urethan dose was increased by the animals' receiving multiple injections of the standard 1 mg/g body weight dose, the same strain-dependence on adenoma induction was shown as that noted when BHT was used as the promoter [table 4]. The number of tumors was increased several times in BALB mice, A-strain mice were stimulated by about 50%, and no effect was observed in the three resistant strains. These results suggest that whatever mechanism regulates adenoma susceptibility, neither repeated dosing with a complete carcinogen nor treatment with a promoter can overcome complete genetic resistance. Tumor incidence and tumor number could be increased by either procedure in the
10
20
NUMBER OF TUMORS
TEXT-FIGURE 2.-Frequency of lung tumors after 1 mg urethan/g body wt
treatment in sensitive SWR/J mice and [SWR X BALB]Fprogeny. Ten SWR mice and 25 [SWR X BALB]F, mice were used.
Strain-Dependent Enhancement of Tumor Number by Multiple BHT or Multiple Urethan Treatments
One approach for the elucidation of the mechanism by which the BALB gene regulates susceptibility to adenoma induction is to test whether it predominantly affects the initiation of the neoplastic process or whether it influences those factors which can be modulated by tumor-promoting agents. BHT causes lung toxicity when injected ip [23]. BHT also can inhibit adenoma formation if administered simultaneously with urethan [5], or it can increase the number of adenomas formed when it is given as repeated ip injections following a single urethan dose [4, 5, 18]. It is probable that metabolites of BHT, rather than BHT itself, regulate these effects [24, 25] and that different metabolites are involved in each process [25]. The capacity of BHT to promote adenomas in one adenoma-sensitive strain [A], the intermediately adenoma-sensitive BALB strain, and the three adenoma-resistant strains was tested [table 3]. BHT increased tumor number threefold to fourfold in BALB mice and by 50% in A-mice. Over the range of BHT concentrations tested, no increase in tumor multiplicity with increasing BHT concentrations was observed. BHT treatment did
Table 3.-Effects of multiple injections of BHT on the promotion of urethan-induced lung adenoma in inbred strains
Treatment [mg/kg Mortality: No. of mice dead/No. of Incidence: No. mice with tumors/total Mean tumor Mouse strain body wt]" mice treated No. of surviving mice
No.ASEM A/J Corn oil 0/12
12/12
22.3+1.47 BHT [400] 0/11
11/11
31.1+2.51 BALB/cByJ Corn oil 0/11
9/11
1.36+0.36 BHT [150] 1/22
21/21
5.0+0.726 BHT [300] 6/23
17/17
4.7120.45 BHT [400] 15/38
22/23
3.26+0.51% C57BL/6J Corn oil 0/5
3/5
0.60+0.24 BHT [400] 5/18
7/13
0.62+0.18 DBA/2J Corn oil 0/5
1/6
0.17 +0.17 BHT [400] 1/20
3/19
0.26+0.15 C3H/2IBG Corn oil 0/5
1/6
0.17+0.17 BHT [400] 10/20
0/10
0
Mice were given injections of 1 mg urethan/g body wt, followed by six weekly injections of either BHT or the corn oil vehicle.
P6 mo after the initial NBU treatment. "Tumors include both neoplastic nodules and hepatocellular carcinomas. dA 5.0-mg DES pellet was grafted subcutaneously on the back and replaced every 2 mo.
* Rats were given 5.0 mg NBU/day in drinking water for 30 days when 50-55 days of age.
Rats received daily sc injections of CB-154 [0.4 mg/100 g body wt] four times/wk throughout the experiment.
TEXT-FIGURE 2.—Body weights and weights of pituitary glands and livers in DES-treated and castrated male WF rats given no further treatment, given
NBU, or given NBU and CB-154 [groups III, IV, and V]. Pituitary weight in castrated male rats given no further treatment or rats given NBU treatment alone [groups I and II]. o, absolute; -, relative.
did not develop PT, the mean weight of the pituitary gland was not different from the mean weights in both rats given no further treatment [group I] and rats given NBU alone [group II]. The mean body weights were not significantly different among groups III, IV, and V.
Histologic observation of HT.-Histologic examination of hepatocellular lesions was diagnosed by the recent recommended classification [18], which is summarized in table 3. Most of the rats that had hepatocellular carcinoma also had neoplastic nodules and foci of cellular alteration [figs. 1-3]. If a rat had these 3 lesions concurrently, we counted this rat
sizes on the surface of the liver. The number of HT increased in rats given DES and NBU [group III]. There was no significant difference in both number of and distribution of HT of various sizes between rats given DES and NBU [group IV] and rats further treated with CB-154 [group V]. Therefore, growth of HT was not affected by treatment with CB-154.
Serum prolactin levels.—Changes of serum prolactin levels in groups III, IV, and V during the 12 months after initial NBU treatment are shown in text-figure 1. The levels of serum prolactin in rats treated with CB-154 [group V] were markedly lower throughout the experiment than were those in the other 2 groups [groups III and IV] and were mostly below about 200 ng/ml. A few samples at 10 and 12 months in group V showed the level above that; these samples were collected from the rats in whom PT was discovered at autopsy. Meanwhile, prolactin levels in groups III and IV were almost above 200 ng/ml and increased sharply about 6 months after the initial NBU treatment. These elevations during the late stage of the experiment appear to be associated with the development of PT. CB-154 treatment to rats given DES and NBU significantly affected the serum prolactin levels.
Liver and pituitary weights.—The absolute or relative weights of livers and pituitary glands in each group are shown in text-figure 2. In rats given DES and NBU [group IV], the mean liver and pituitary weights were increased when compared with those in rats given DES alone [group III]. The mean liver weights were not different between rats given DES and NBU [group IV] and rats further treated with CB-154 [group V], although the mean pituitary weight was reduced by about 74% in group V when compared with that in group IV. Furthermore, in 16 rats of group V that
as having hepatocellular carcinoma as described previously in [14]. Inasmuch as neoplastic nodules have been considered to be neoplastic in nature [18], we interpreted hepatocellular carcinoma and neoplastic nodules as HT. In groups III and IV, neoplastic nodules and hepatocellular carcinoma were 3 [33%] and 1 [11%] and 4 [24%] and 11 [65%], respectively. The highly differentiated HT was observed in rats given DES alone [group III] more thar it was in rats given DES and NBU [group IV]. The distribution of histologic classification in rats further treated with CB-154 [group V] did not differ significantly from that observed in rats given DES and NBU [group IV].
No metastasis formation was detected in any HT case, and no HT was associated with cirrhosis. Furthermore, about 50% of the rats with HT showed a positive reaction for a-fetoprotein [14], whereas none of the rats had neoplastic lesions of bile duct origin.
Histologic observation of PT.-The glands weighing 30 mg, as proposed by Clifton and Meyer [19], or macroscopically visible tumors, which are blood-rich, dark red, and elastic soft, were interpreted to be PT [fig. 4]. Microscopically, they fell within the classification of hemorrhagic chromophobe adenoma; tumor cells were round and medium size with pale nuclei, and cytoplasms were weak eosinophilic or chromophobic. Mammary glands of PT-bearing rats were hyperplastic when the milk secretion was found [14]. The development of MT, most of which were adenocarcinomas, was associated with the presence of PT.
Prolactin levels in groups III and IV increased sharply 6 months after the initial NBU treatment. These elevations of serum prolactin levels apparently were correlated with the development of PT whose incidence was 7/9 [78%] in group III and 12/17 [71%] in group IV. In group V serum prolactin levels were mostly below the 200 ng/ml level and were markedly lower than those in groups III and IV throughout the experiment, although a few samples showed the next higher level. These samples with the high serum prolactin level at 10 and 12 months were collected from rats that developed PT, which was recognized at autopsy [group V]. CB-154 treatment of rats given DES and NBU significantly reduced not only the incidence of PT from 71 to 20% but also reduced the mean weight of the pituitary gland from 57.4 to 14.7 mg. Furthermore, in 16 rats that did not develop PT [group V], pituitary gland weight was reduced as compared with that in both rats given no further treatment [group I] and rats given NBU treatment [group II]. Thus CB-154 was considered effective in the suppression of PT development and growth and in the suppression of prolactin secretion. On the contrary, CB-154 was ineffective in the reduction of the incidence of HT, the mean number of HT, and the mean weight of liver, and CB-154 did not alter the distribution of the histologic classification of hepatocellular lesions [group V].
Moribund or dead rats during the late stage of the experiment had already developed HT when they were completely autopsied, and the serum prolactin levels from these rats were low [group V]. The development of HT was considered not to be associated with the prolactin level. Therefore, prolactin does not seem to play a key role in the hepatic tumorigenesis in this study. We have had the belief that DES action on the liver cell is not mediated via the pituitary gland in the secretion of prolactin, but we do not contradict that other pituitary hormones may be able to affect the liver cell tumor formation. Recent reports have shown that estrogen, prolactin, and growth hormone bound specifically to the livers of several species, indicating the presence of receptors for each hormone on the liver [21-23]. In general, implantation of a DES pellet induces the development of PT that possess mammosomatotropin [5, 26], and these hormones have been shown to promote the chemical carcinogen-induced hepatocarcinogenesis in rats [25, 26]. Our previous study also showed that grafted mammotropic hormone-secreting PT, originally induced by DES, that have been transplanted in the syngeneic rat shortened the latent period of HT in castrated male rats given DES and NBU, particularly during the early stage of the experiment, but they did not increase the incidence of HT at the termination of the experiment [14]. Furthermore, hypophysectomy or lesions in the median eminence area of the hypothalamus inhibited the hepatic carcinogenicity of several powerful carcinogens [27, 28]. Thus growth hormone might be the main important pituitary hormone responsible for liver tumor induction by chemical carcinogens, although its effect might be considered not significant in this study.
The findings of the present study suggest that DES may act directly on the liver cell as a weak carcinogen. This supposition is supported by our recent observation that the prolonged administration of antiestrogens inhibit the development of HT in castrated male rats treated with DES and
In the present study we investigated whether DES itself has a direct carcinogenic effect on the livers of castrated male rats given DES and NBU. About 44% of the castrated male rats given DES alone developed HT [group III]. This result is evidence that HT were induced by DES; this supports the report of Reznik-Schüller [4] that 29% of the European hamsters treated with subcutaneous implants of DES pellets developed liver tumors. In addition, further treatment by NBU in these DES-treated rats increased the incidence of HT two times greater than it did in rats without NBU treatment [group III], indicating that the development of HT was significantly elicited by the subthreshold dose of NBU that, even in female rats, could not induce the tumorigenesis of the mammary gland as primary target tissue [12]. The liver has not been considered to be the target tissue of NBU [12, 15]. Therefore, a small number of chemical agents such as NBU in this study, which has appeared noncarcinogenic, might have contributed to the induction of the tumor formation when the hormone environment in the host is modified by exogenous hormones.
The interaction of NBU and DES on the metabolic activation or the detoxification is not obvious in this study. However, NBU as well as irradiation [14] appears to have an initiating effect. Takizawa and Nishihara [20] pointed out the multicarcinogenicity of NBU in regard to its spectrum of target organ. NBU also might have promoted a initiating effect of DES by acting as a preconditioning factor and/or as an immunosuppressor. In general, chemical carcinogens have been known to suppress the host immunologic competence.
NBU [Sumi C: Unpublished data]. DES appears to act in both the initiation and promotion processes of HT formation. As discussed previously [14], DES also might have played a role as a conditioning factor; its sustained stimuli on the male liver cells conceivably caused chronic cell injury, which rendered the cells more susceptible to the carcinogens such as NBU or to irradiation. Estrogens have been reported to cause hepatic dysfunction with depression of hepatic transport systems for organic anions [29], and the metabolic activation of DES and its possible relation to carcinogenic effect have been recently investigated [30, 31].
The carcinogenic effect of DES might have been enhanced by the castration. We have recently found that none of the intact male or female rats developed HT when DES was administrated, whereas castrated female rats had a low incidence of HT by the same treatment [Sumi C: Unpublished data]. The condition of being castrated might not only negate counteraction by the male sex hormones to DES but also might cause serious changes in the overall hormone balance of host rats.
The increased development of HT in our system was primarily unexpected because, in general and as induced by various chemicals, rat liver carcinogenesis has been well known to be enhanced by the male sex hormones [16]. However, our findings may be relevant to the recent observation of liver cell adenoma associated with the use of oral contraceptives [2, 6-8]. Since the paper of Baum et al. [8], it has been known that the incidence of hepatocellular tumor development increased among women under the influence of oral contraceptives. Inasmuch as the incidence of primary HT in individuals untreated with sex hormones is rare in Europe and the United States, an association has been suggested between the use of oral contraceptives and the development of primary HT in women. However, the carcinogenicity of oral contraceptives on the liver was concluded to be negative by the Committee on Safety of Medicine [3] because long-term treatment of rats with contraceptives did not increase the incidence of hepatomas. Therefore, the high yield in rats of HT that was produced in our experimental system is considered to be a useful tool for the study of the mechanism of the HT associated with steroids and for the detection of the carcinogenicity of unknown chemical agents that act synergistically with these steroids on the liver. Further investigations are required.
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Figure 1.—Gross appearance of HT in castrated male WF rats given DES alone, diagnosed histologically as hepatocellular carcinoma. Large arrow, HT:
double-headed arrow, right kidney; small arrow, spleen. FIGURE 2.-Histologic appearance of foci of cellular alteration in castrated male WF rats given DES and NBU. Foci are compressing the surrounding liver
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proliferation of tumor cells in dilated sinusoid. H&E. Bar=25 um. FIGURE 4.—Gross appearance of PT in castrated male WF rats given DES and NBU; gland weighs 187 mg. Huge tumor mass, dark red and elastic soft,
Page 6
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Figure 1.-Morphology of cells treated with RA. A] Confluent culture of cuboidal Rama 25 cells in BM for 2 days. B] Confluent
culture of criss-cross elongated Rama 521 cells treated with 3.3 uM RA and BM for 2 days. C] Confluent culture of Rama 259 cells treated for 2 days with BM and RA. D] Confluent culture of Rama 25 cells treated for 2 days with BM and RA showing droplet cells and a dome [D]. A-D] Living cultures were photographed with phase-contrast optics. White bar=100 um. X 190
n-propylamine Derivatives 1,2,3
W. Lijinsky,4 M. D. Reuber, 4 J. E. Saavedra, 4 and G. M. Singer 4
compound has been identified as a metabolite of several Nnitrosomethyl-n-alkylamines [5] that gave rise to tumors of the bladder in rats when given by gavage [1], and the concentration of the gavage solution [63 mg/ml] was equimolar with that of the nitrosomethylalkylamine treatments. N-Nitrosomethyl[3-carboxypropyl]amine, also a urinary metabolite of several N-nitrosomethyl-n-alkylamines having an even number of carbons in the chain, was administered as a 2-mM solution because nitrosamino acids are usually weak carcinogens. The concentrations of compound in the various treatment groups are given in table 1.
ABSTRACT — The carcinogenicity of N-nitrosomethyl-n-propylamine and five of its derivatives, including N-nitrosomethyl-n-butylamine, was compared by oral administration of the compounds to inbred F344 rats. N-Nitromethyl-n-propylamine and N-nitrosomethyl-n-butylamine given in drinking water induced tumors of the upper gastrointestinal tract, mainly carcinomas of the esophagus, and appeared to be of comparable potency. N-Nitrosomethyl[2-hydroxypropyl]amine also mainly induced esophageal carcinomas [100% incidence] and lung tumors, whereas N-nitrosomethyl[2,3-dihydroxypropyl]amine mainly induced nasal cavity tumors and gave rise to a high incidence of esophageal tumors; however, it appeared to be less potent than the monohydroxy compound. N-Nitrosomethyl[2-oxopropyl]amine, the ketone corresponding to N-nitrosomethyl[2-hydroxypropyl]amine, was a more potent carcinogen than the latter at comparable doses in drinking water and gave rise to a high incidence of esophageal tumors and tumors of the trachea; female rats had a high incidence [15/20] of angiosarcomas of the liver, but only 2 male rats died with this tumor. When N-nitrosomethyl[2-oxopropyl]amine was administered at a lower dose in drinking water or at the same dose given by gavage, the incidence of esophageal tumors was lower and there were fewer carcinomas. After administration of large doses in drinking water to male and female rats, N-nitrosomethyl[3-carboxypropyl]amine, a urinary metabolite of several N-nitrosomethyl-n-alkylamines that induce tumors of the urinary bladder in rats, gave rise to a high incidence of transitional cell carcinomas of the bladder. The time to death of animals with these tumors was long, and there were few other tumors.—JNCI 1983; 70:959-963.
Chemicals.-N-Nitrosomethyl-n-propylamine and N-nitrosomethyl-n-butylamine were prepared by nitrosation of the corresponding amines [Aldrich Chemical Co., Milwaukee, Wis.] and were distilled under reduced pressure. Their mass spectra and NMR spectra showed them to be of high purity. N-nitrosomethyl[2-hydroxypropyl]amine, N-nitrosomethyl[2-oxopropyl]amine, and N-nitrosomethyl[3-carboxypropyl]amine were prepared as described by Singer et al. [5].
N-Nitrosomethyl[2,3-dihydroxypropyl]amine. First N-nitrosomethyl[2,3-dihydroxypropylamine was prepared by the addition of 26 ml [0.5 mol] of glycidol in 50 ml methanol to 60 g [1.9 mol] of methylamine in 500 ml methanol at 0°C, after which the solution was stirred at room temperature for 4 hours. The methanol and excess methylamine were removed on a rotary evaporator, and the residue was vacuum distilled to give 25 g [46%] of the amine [boiling point, 856°C at 0.1 mm Hg; infrared spectrophotometry [film], 3,360, 2,940, 1,460, 1,080, 1,040, and 860 cm NMR [CDC13], 8 2.42 [s, 3H], 2.66 [d, 2H], and 3.4–3.91 [m, 5H];MS, m/z [%], 106 [M* + 1, 17], 105 [M", 2], 88 [4], 87 [22], 86 [3], 75 [6], 74 [100], 70 [9], 59 [12], 58 [21], and 56 [43]].
For the preparation of the nitroso derivative, a solution of 19.5 g [0.186 mol] of N-methyl[2,3-dihydroxypropyl]amine
A number of N-nitrosomethyl-n-alkylamines containing alkyl chains of different lengths were administered to rats by gavage, and their carcinogenic effectiveness was compared [1]. Similarly, the effects on carcinogenicity of various substituents in the ethyl group of N-nitrosomethylethylamine have been investigated [2]. In the latter study, the considerable influence of the substituents on the potency of the resulting nitrosamine led us to study derivatives of Nnitrosomethyl-n-propylamine. Again, the objective was similar-to gain insight into the relative importance of oxidation of the methyl group and of the substituted n-propyl group in carcinogenesis. This information can then be compared with the results of in vitro studies of metabolism by rat liver microsomes [3, 4].
The derivatives of N-nitrosomethyl-n-propylamine [textfig. 1] used in this study include those with one or more hydroxyl groups, a methyl group, a carbonyl group, or a carboxyl group in the propyl chain. It was originally planned to administer all of the compounds in drinking water at equimolar concentrations to make comparisons of potency easier. However, because of their great toxicity, Nnitrosomethyl-n-propylamine and N-nitrosomethyl-n-butylamine were given at low concentrations. One compound, N-nitrosomethyl[2-oxopropyl]amine, was administered to rats by gavage as well as in the drinking water. This
ABBREVIATIONS USED: MS=mass spectrometry; m/z=mass/charge; NMR=nuclear magnetic resonance.
Received September 7, 1982; accepted December 7, 1982.
Supported by Public Health Service contract NOICO-75380 from the National Cancer Institute. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
methanol to give 14 g [56%] N-nitrosomethyl[2,3-dihydroxypropyl]amine [infrared spectrophotometry [film], 3,380, 2,920, 1,440, 1,420, 1,330, and 1,030 cm-?; NMR [acetonedo], 8 3.12 [s, 2.45H] syn CH3, 3.88 [s, 0.55H] anti-CH3, 3.63 [d, 2H], and 3.44-4.12 [complex, 5H]; UV spectrophotometry, Imax [€], 338 [97]; MS, m/2 [%], 135 [1.8], 134 [M*, 0.5], 116 [2], 105 [1.4], 104 [0.9], 103 [3], 87 [5], 86 [3], 75 [3], 74 [15], 73 [46], 72 [9], 61 [100], 60 [5], 59 [8], 58 [17]. 57 [10], and 56 [11]]. Analysis: C4H10N203; calculated: C, 35.82; H, 7.51; N, 20.89
found: C, 35.70; H, 7.63; N, 21.04
in 180 ml water at 5°C was acidified with 24 ml concentrated hydrochloric acid. To the resulting solution was added 33 g [0.48 mol] sodium nitrite in small lots over 15 minutes. The ice bath was removed, and the reaction mixture was stirred at 25°C for 2 hours. The product was extracted into ethyl acetate, which was then evaporated leaving a yellow oil. The crude material was treated with methanol-HCl until evolution of methyl nitrite ceased. The material was reextracted into ethyl acetate, washed with 5% sodium bicarbonate solution, and dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator, and the product was chromatographed through dry-packed silica gel and eluted with 10:1 chloroform:
Animal treatments.—A group of 20 F344 rats 7-8 weeks old from the colony of the NCI-Frederick Cancer Research Facility, bred and maintained in a barrier facility, was used for each treatment. These animals display nearly uniform weights and growth characteristics, adult males weighing approximately 400 g and females weighing approximately 200 g. The animals were housed 4 to a plastic cage with a wire-mesh bottom and fed Rockland rat diet in pellets ad libitum. For those groups receiving the nitrosamines in drinking water, a solution of the nitrosamine was given a the rate of 20 ml per rat per day, 5 days a week; all of the solution was consumed with little spillage. So that water deficiency could be avoided, on the other 2 days of each week tap water was given ad libitum, as it was continuously to those groups in which treatment was by gavage. All six compounds were given to female rats, but groups of males
used for N-nitrosomethyl[3-carboxypropyl]amine treatment and for three types of treatment with N-nitroso
Oropharynx 7 [5] Tongue 3 [2] Epiglottis 3 [2] Oropharynx 8 [5] Tongue 2 [2] Leukemia 2 Trachea 6 [0] Bronchus carcinoma 1 Trachea 11 [10] Kidney pelvis carcinoma 1 Pancreas acinar cell adenoma 1 Tongue 4 [2] Trachea 2 [1] Leukemia 9 Lung adenocarcinoma 8 Lung squamous cell carcinoma 7 Trachea 1 [0] Tongue 1 [1] Epiglottis 1 [1] Kidney interstitial cell sarcoma 1 Tongue 3 [2] Lung 2 [1] Pituitary gland 3 [2] Uterus carcinoma 1 Bladder polyp 1
methyl[2-oxopropyl]amine. For economic reasons both sexes were not used for all treatments. The treatments lasted a prescribed number of weeks, after which time the animals were observed until natural death. Each animal was necropsied. All lesions and major organs were fixed in Formalin for microscopic examination. No special group of untreated controls was kept, since groups of untreated F344 rats are continuously maintained in the facility. Contemporary control animals did not begin to die until the 80th week; there was 90% survival at 100 weeks.
The survival times among treated animals are shown in table 1. In all groups except those given N-nitrosomethyl[3carboxypropyl]amine, most of the rats were dead by week 50, and the cause of death was almost invariably tumors induced by the treatments; tumors of the esophagus, liver, and nasal cavity were the most common tumors. The number of animals with each type of tumor is also given in table 1. The bladder tumors induced by N-nitrosomethyl[3-carboxypropyl]amine took considerably longer to kill the animals.
The mortality rates of the rats treated with N-nitrosomethyl-n-propylamine and its higher homologue N-nitrosomethyl-n-butylamine, which were administered at approximately equimolar doses totaling 0.3 mmol, were similar. This result suggests that these two compounds are of similar carcinogenic potency, since, in both cases, the rats died of tumors of the upper gastrointestinal tract, mainly carcinomas of the esophagus.
By the same criterion the oxygenated N-nitrosomethyl-n
propylamines were considerably less potent than was the parent compound. Again, the common cause of death in these groups was neoplasms of the esophagus, but a smaller dose of N-nitrosomethyl-n-propylamine led to death of the animals almost as rapidly as did the derivatives. N-Nitrosomethyl[2-hydroxypropyl]amine and N-nitrosomethyl[2,3dihydroxypropyl]amine were much less potent by this criterion than was N-nitrosomethyl-n-propylamine, whereas Nnitrosomethyl[2-oxopropyl]amine was only somewhat less potent. The mortality rates of male and female rats given 100 mg N-nitrosomethyl[2-oxopropyl]amine per liter were similar, but 15 females had angiosarcomas of the liver compared with 2 of the males.
Tumors of the nasal cavity [squamous cell carcinomas and olfactory adenocarcinomas] and of the trachea were common in the rats treated with N-nitrosomethyl[2-oxopropyl]amine and N-nitrosomethyl[2,3-dihydroxypropyl]amine, but they were rare or absent in other groups. Animals given N-nitrosomethyl[2-hydroxypropyl]amine had a high incicence of lung tumors [squamous cell carcinomas and adenocarcinomas] not seen in the other groups. In contrast, the rats treated with N-nitrosomethyl[3-carboxypropyl]amine, although they survived much better than any of the other groups, had almost exclusively transitional cell carcinomas of the urinary bladder, the only exceptions being 2 females with liver tumors as well.
It appears that the carcinogenic potency of N-nitrosomethyl-n-alkylamines increases considerably with increasing chain length from -ethyl to -n-propyl, but an additional
Page 9
carbon atom in the chain, as in N-nitrosomethyl-n-butylamine, did not further increase potency. However, as measured by the rate of mortality from tumors, all of the other substituents in the propyl group decreased the carcinogenic potency. In the case of the 2-hydroxypropyl derivative, the carcinogenic potency was considerably reduced. Rats treated with six times the dose of N-nitrosomethyl[2-hydroxypropyl]amine died at the same rate from esophageal tumors as did the group treated with N-nitrosomethyl-n-propylamine. The corresponding ketone N-nitrosomethyl[2-oxopropyl]amine was somewhat more potent than the alcohol, but it was less potent than the unsubstituted propyl compound. Animals treated with N-nitrosomethyl[2-oxopropyl]amine had a lower mortality rate from esophageal tumors than those receiving half that dose of N-nitrosomethyl-n-propylamine. The mortality rate of rats treated with six times the dose of the ketone was greater than that of rats treated with the unsubtituted propyl compound. N-Nitrosomethyl[2-oxopropyl]amine is partially reduced in vivo to the alcohol Nnitrosomethyl[2-hydroxypropyl]amine, which appears in the urine [5].
The rats receiving the 2-oxopropyl compound by gavage suffered considerable toxicity; 5 rats died or were moribund after 8 weeks of treatment, which was then stopped. Although these animals had then received a dose comparable with the dose [100 mg/liter] given in drinking water, the numbers of animals with malignant tumors of the upper gastrointestinal tract and nasal cavity were smaller. The rats in this group developed severe pericentral necrosis of the liver with severe acute hemorrhage, followed by hemorrhage in many
other
organs of the body, such as the thymus, lymph nodes, and meninges, as well as old and recent hemorrhage into the gastrointestinal tract. Most of these rats did not survive long enough to develop many neoplasms, but several had papillomas of the esophagus or early monocytic leukemia, the latter being common in old untreated F344 rats. It can be assumed that the periodic treatment with larger doses of this nitrosoamine is less effective than are continuous smaller doses given in drinking water.
There were no bladder tumors in the rats treated with Nnitrosomethyl[2-oxopropyl]amine, which suggests that despite the presence of this metabolite in the urine of rats treated with even-numbered long-chain N-nitrosomethyl-nalkylamines, this metabolite is not responsible for the bladder tumors induced by these compounds [1]. Since the longchain N-nitrosomethyl-n-alkylamines above octyl- did not induce tumors of the upper gastrointestinal tract, the extent of formation of N-nitrosomethyl[2-oxopropyl]amine from them in vivo must be insufficient to induce those tumors. There is evidence that nitrosamines can induce tumors of the upper gastrointestinal tract systemically and not necessarily through direct contact [6].
N-Nitrosomethyl[2-oxopropyl]amine at 100 mg/liter in drinking water induced a high incidence [15/20] of angiosarcomas of the liver in female rats; only 2 male rats given this treatment developed liver tumors. This seems to be a rare case of one sex of rats being susceptible to a particular nitrosamine-induced tumor, while the other sex is not. However, the other treatments with this compound [25 mg/liter in water and 63 mg/ml in oil] were given only to males,
which did not develop liver tumors, and it is not known whether liver tumors would have developed in female rats given those treatments. Possibly, the higher dose per unit body weight received by the females induced greater liver toxicity and consequently more liver tumors. In contrast, this compound given by sc injection into Syrian hamsters was reported to give rise to tumors of the nasal cavity, pancreas, liver, and kidneys [7].
N-Nitrosomethyl[3-carboxypropyl]amine is a common urinary metabolite of N-nitrosomethyl-n-alkylamines containing even numbers of carbons in the chain [5]. These nitrosamines, other than butyl- and hexyl-, have regularly induced transitional cell carcinomas of the urinary bladder and, in most cases, these were the only tumors induced in male rats [1]. Our finding that N-nitrosomethyl[3-carboxypropyl]amine is a bladder carcinogen in rats seems to support the suggestion of Okada et al. [8] that this nitrosamino acid is the proximate carcinogenic metabolite of those Nnitrosomethyl-n-alkylamines. This could be true, but the doses of the nitrosamino acid received by each rat in these experiments were considerably higher than those of the Vnitrosomethyl-n-alkylamines that gave rise to bladder tumors. Even if the conversion of the latter to the nitrosamino aci were 100% [and it was not, since other metabolites were found in the urine [5]], the N-nitrosomethyl[3-carboxypropyl]amine would seem to be insufficiently potent to be considered the proximate carcinogen. The pharmacology of these compounds has not, however, been studied in depth, and the explanation for the discrepancy might lie in the pharmacodynamics of the compounds. It is equally possible that N-nitrosomethyl[3-carboxypropyl]amine, which is itself metabolized in rats [5], might have in common with the Nnitrosomethyl-n-alkylamines a metabolite that is the proximate carcinogen. The N-nitrosomethyl-n-alkylamines are usually mutagenic to bacteria [9], whereas N-nitrosomethyl[3-carboxypropyl]amine is nonmutagenic to Salmonella tiphimurium [Andrews AW, Lijinsky W: Unpublished observations].
It is not clear why the rat esophagus is so common a target for the derivatives of N-nitrosomethyl-n-propylamine examined. It is clear, however, that substitution in the propyl group decreased carcinogenic effectiveness, which is not supportive of the idea of Krüger that ß-oxidation of the propyl group was an important step in activation of propylnitrosamines [10]. The present results are consistent with those of Blattmann and Preussmann [11] and others who found that oxidation of nitrosamines can take place at many sites on the molecule other than the a-carbon atoms. These alternative oxidations can possibly lead to reduction in effectiveness due, for example, to easier excretion. This could be the reason for the lower effectiveness of N-nitrosomethyl[2-hydroxypropyl]amine and the still lower effectiveness of N-nitrosomethyl[2,3-dihydroxypropyl]amine.
These compounds could all act by methylation of DNA in the rat esophagus, as suggested by Hodgson et al. [12] Such methylation would require formation of a methylating agent from the nitrosamines by oxidation of the propyl group at the a-position, forming an aldehyde, which in all of these cases is a common product of intermediary metabolism, pyruvaldehyde, lactaldehyde, or glyceraldehyde.
However, oxidation of the methyl group of these substituted propylnitrosamines would release activated forms of these aldehydes, and the effect of these forms is unknown. Formaldehyde is commonly formed by incubation of N-nitrosomethyl-n-alkylamines of this type with rat liver microsomes, but the aldehyde derived from oxidation of the other side of the molecule is formed in much smaller amounts, and sometimes it is not detected at all [Farrelly JG: Unpublished observations]. The precise mechanism by which N-nitrosomethyl-n-alkylamines induce esophageal tumors in rats is unknown. Unfortunately, studies of metabolic activation in the esophagus and bladder of the rat are made difficult by the sparseness of these tissues.
nitrosomethyl-n-butylamine and nitrosomethylphenylethylamine.
Cancer Res 1982; 42:2105-2109. [5] SINGER GM, LIJINSKY W, BUETTNER L, McClusKY GA. Relationship
of rat urinary metabolites of N-nitrosomethyl-n-alkylamine to blad
der carcinogenesis. Cancer Res 1981; 41:4942-4946. [6] DRUCKREY H, PREUSSMANN R, IVANKOVIC S, SCHMÄHL D. Organotrope
carcinogene Wirkungen bei 65 Verschiedenen N-Nitroso-Verbin
dungen an BD-Ratten. Z Krebsforsch 1967; 69:103-201. [7] Pour P, GIngelL R, LANGENBACH R, et al. Carcinogenicity of N
nitrosomethyl[2-oxopropyl]amine in Syrian hamsters. Cancer Res
1980; 40:3585-3590. [8] OKADA M, Suzuki E, Mochizuki M. Possible important role of
urinary N-methyl-N[3-carboxypropyl] nitrosamine in the induction of bladder tumors in rats by N-methyl-N-dodecylnitrosamine. Gan
1976; 67:771-772. [9] ANDREWS AW, LIJINSKY W. Mutagenicity of 45 nitrosamines in Sal
[1] LIJINSKY W, SAAVEDRA J, REUBER MD. Induction of carcinogenesis
ABSTRACT—Friend erythroleukemia cells [FL cells] derived from DBA/2 mice may be induced to differentiate in vitro by addition of dimethyl sulfoxide [DMSO] to the culture medium. Transglutaminase [glutaminyl-peptide y-glutamyltransferase, EC 2.3.2.13] [TGase] activity was detected in the lysates of uninduced FL cells and was markedly increased in DMSO-treated cells. DMSO induced TGase activity of differentiating FL cells in a dose-dependent manner over the concentration range 0-280 mm. The increase in TGase activity was observed after 1 day's cultivation of the cells with 280 MM DMSO and continued to 4 days. Another differentiation inducing agent, butyric acid [1.4 MM], was as effective as DMSO in enhancing TGase activity in FL cells. Treatment of controls, a lymphoma cell line and mouse erythrocytes, with DMSO or butyric acid had no effect. These results suggest that the increase in TGase activity in DMSO-or butyric acid-treated FL cells may be related to cell differentiation.- JNCI 1983; 70:965-969.
When grown in tissue culture, FL cells may be induced to differentiate along the erythroid pathway by addition of DMSO [1] to the growth medium or by various other agents [2]. Treatment of FL cells with inducing agents leads to a gradual arrest of cell division [3-5], which is accompanied by chromatin condensation [6] and a decrease in cell volume [7]. Several biochemical markers have been identified after induction, including the accumulation of globin mRNA [813] and globin chain synthesis [14, 15], increases in heme synthesis [6, 16], synthesis of carbonic anhydrase [17], appearance of erythrocyte-specific membrane antigens [18, 19], and membrane proteins such as spectrin [20, 21], and
YAC-1, a lymphoma cell line, was used as described elsewhere [27]. Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum [Reheis Chemical Co., Kankakee, III.], 100 U penicillin/ml and 100 ug streptomycin/ml [Flow Laboratories, Rockville, Md.] at 37°C in a 95% air-5% CO2 atmosphere. Cells were plated at 1X10%/ ml. Recultured cells or cells treated with DMSO or butyric acid [Sigma Chemical Co., St. Louis, Mo.] were obtained from previous culture of exponentially growing cells.
TGase activity assay. — TGase was assayed essentially as described [28] by incorporating [H]putrescine [New England Nuclear Corp., Boston, Mass.] into N,N-dimethylated casein [Calbiochem-Behring Corp., La Jolla, Cal.] in the presence of Ca2+. The cell suspensions were washed once with 50 mM Tris-HCl buffer [pH 7.5] containing 0.15 M NaCl. The pellet was resuspended in the same buffer at a final concentration of 5.0x10' cells/ml and then frozen and thawed twice. The assay mixture [at 37°] consisted of 10 ul CaCl2 [50 mm], 20 ul N,N'-dimethylated casein [2%], 10 ul [H]putrescine [12 mM, sp act 2 uCi/120 nmol], and 40 ul cell lysate. After various times [0-90 min], 10-ul aliquots were placed on 1-cm? Whatman 3MM filter paper and treated with 10% trichloroacetic acid. The filter papers were washed with 5% trichloroacetic acid followed by an ethanolacetone [1:1] mixture and then by acetone, and the radioactivity of the insoluble material was measured in a Packard liquid scintillation counter.
We calculated the specific activity of TGase by subtracting the zero-time control from the experimental value. One unit of enzyme activity is defined as nanomoles of putrescine incorporated per 30 minutes with 0.4 mg N,N'-dimethylated casein as acceptor substrate.
Protein determination. — The procedure of the Bio-Rad protein assay was used. The principle of this dye-binding method was first described by Bradford [29].
Hemoglobin determination.—Cells were collected and washed with a phosphate-buffered saline solution [pH 7.5] and lysed by freezing and thawing 5x10' cells/ml. Following centrifugation at 20,000Xg for 30 minutes, the supernatant was collected and assayed for hemoglobin content as described [30]. The 20 ul supernatant was mixed with 0.2 ml 1% benzidine solution in 90% acetic acid and 0.2 ml freshly prepared 1% hydrogen peroxide. After incubation at room temperature for 20 minutes, 2 ml 10% acetic acid was added
TGase [glutaminyl-peptide Y-glutamyltransferase, EC 2.3.2.13] catalyzes an acyl transfer reaction between the ycarboxamide groups of protein-bound glutamine and primary amino
groups.
When the amine is the e-amino residue of peptide-bound lysine, intermolecular or intramolecular €-[y-glutamyl] lysine isopeptide cross-links are formed. TGase enzymes exist widely in nature and have been shown to perform several biologically important functions, i.e., the stabilization of the fibrin structure during blood coagulation [23] and the production of the vaginal plug by postejaculatory clotting of rodent seminal plasma [24]. Work on human erythrocytes [25, 26] has indicated that TGase plays a role in Ca2+-mediated cross-linking of erythrocyte membrane proteins. The studies reported here demonstrate a marked increase in TGase activity during erythroid differentiation of FL cells. This phenomenon may be related to stabilization of cell membranes by cross-linking membrane proteins.
ABBREVIATIONS USED: DMSO=dimethyl sulfoxide; FL=Friend erythroleukemia; TGase=transglutaminase [glutaminyl-peptide y-glutamyltransferase, EC 2.3.2.13].
Cell lines. —GM 979, a Friend virus-infected leukemia cell line derived from DBA/2 ice, was obtained from G. H. Lyman, University of South Florida College of Medicine.
Received September 10, 1982; accepted December 28, 1982.
Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, Tampa, Fla. 33612.
TGase activity was present in cell lysates of cultured FL cell line GM 979 as determined by assaying the incorporation of [H]putrescine into N,N’-dimethylated casein in the presence of Ca2+. The enzyme activity in GM 979 cells at exponential growth was in the range of 4 to 6 U/mg protein. The TGase increased eightfold to fifteenfold in cells cultured for 4 days in medium containing 280 mM DMSO. Enzyme activity was not detected in intact cell preparations but was detected only in cell lysates. Without addition of Ca2+, a time dependent, low level of [H]putrescine incorporation could be detected. However, upon addition of 1 mM EDTA in the assay mixture, the incorporation of [H]putrescine into N,N'-dimethylated casein was no longer detectable.
The ability of several amines to inhibit DMSO-induced TGase activity was examined in GM 979 cells. The synthetic amine, cystamine, has been found capable of inactivating TGase [31]. 1,5-Pentanediamine, histamine, methylamine, and ethylamine, as well as unlabeled, cold putrescine, are known to serve as substrates and compete with [H]putrescine for y-glutamyl acceptor binding sites. As shown in textfigure 1, all of the amines tested inhibited [H]putrescine incorporation. Cystamine was the most potent inhibitor, inhibiting 50% of the total enzyme activity at a concentration of 0.3 mm; 100% inhibition was obtained with a concentration of 5.8 mM. Histamine was as effective as putrescine in inhibiting [H]putrescine incorporation, followed by 1,5-pentanediamine, methylamine, and ethyla
DMSO [ mm ] TEXT-FIGURE 2.– TGase activity in FL cells treated with DMSO at differ
ent concentrations. FL cells were cultured with DMSO at different concentrations for 3 days [O], or 4 days followed by measurement of TGase activity in cell lysates.
mine, which were less active than histamine in competing with putrescine for acceptor binding sites on N.V"-dimethylated casein. DMSO induced TGase activity in a dose-dependent manThe enzyme
activities detected in FL cells treated with DMSO for 3 or 4 days showed a linear relationship with DMSO concentration over the range of 0-280 mM [text-fig. 2]. Four-day treatment resulted in consistently higher enzyme activity than that achieved by 3-day treatment. DMSO concentration higher than 280 mM was not tested because of its toxic effects.
Birckbichler et al. [32] reported that enhanced TGase activity correlates with the nonproliferating state of cultured cells. To distinguish whether the increase in TGase activity in DMSO-treated FL cells was associated with cell differentiation or cell proliferation, we established growth curves of GM 979 in the presence or absence of DMSO and timedependent TGase activities in these cultured cells. As shown in text-figure 3, the increase in TGase activity was observed after 1 day's cultivation of GM 979 in 280 mM DMSO, and it continued to 4 days. During the period of exponential growth, days 1-3 after culturing, enzymatic activity of untreated GM 979 was low and relatively constant. On the 4th day, when cells ceased growing, TGase activity increased 2.3-fold as compared to 1 day before. This observation is consistent with the finding of Birckbichler et al. [32] that TGase activity is increased in nonproliferating cells. However, a 6.5-fold higher activity was found in DMSO-treated cells when compared with untreated, 4-day cultured cells. This result suggests that DMSO-induced TGase activity is more likely associated with cell differentiation than with cell proliferation. This hypothesis was further supported by the finding that another differentiation inducing agent, i.e., butyric acid, was as effective as DMSO in enhancing TGase activity in FL cells. Also, 1.4 mM butyric acid induced GM 979 cell differentiation. Cultivation of GM 979 cells for 3
CONCENTRATION [mm] TEXT-FIGURE 1.-Inhibition of TGase in FL cell lysates. Log dose-response curves for inhibition of DMSO-induced TGase in FL cells by cystamine
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The role of TGase in erythroid differentiation requires further investigation. The continuous increase in TGase activity during exponential growth of DMSO-treated FL cells showed no direct correlation to the nonproliferating state of the cells. However, DMSO- or butyric acid-treated FL cells, which are committed to terminal differentiation, eventually lost their proliferative capacity. It is possible that the higher TGase activity in differentiating FL cells is related to the stabilization of cell membranes by crosslinking membrane proteins which, in turn, leads to an irreversible arrest of cell division. Alternatively, the enzyme's role may be to post modify membrane proteins or other proteins with small metabolites such as polyamines and provide signals leading to the terminal nonproliferating state of the cells. It has been reported that small amounts of putrescine and spermidine are bound covalently to proteins through the catalytic reaction of TGase in human blood lymphocytes after treatment with mitogens [33].
The presence of the erythrocyte membrane-associated protein spectrin in FL cells has been reported. Spectrin increased tenfold to twentyfold after exposure of the cells to DMSO. The intracellular concentration of spectrin reached a peak on the 3d day of DMSO treatment, after which levels fell to those found in mouse erythrocytes [21]. The synthesis of transmembrane glycoprotein band 3 was also reported in FL cells treated with DMSO [22]. A greater than tenfold increase was observed, and maximal synthesis was attained 3-4 days after the beginning of induction. Results from experiments with Ca't-loaded human erythrocytes [26] demonstrated that the Ca2+ can switch on the latent TGase in erythrocytes, which causes the covalent fusion of membrane proteins by y-glutamyl-e-lysine bridges. The formation of membrane protein polymers was accompanied by the disappearance of band 4.1 and a reduction in the amounts of proteins in the spectrin and band 3 regions [26].
The involvement of spectrin and band 3 in TGase-catalyzed reactions in differentiating FL cells is a viable possibility. Further analyses of these complex metabolic changes appear warranted. Nevertheless, it appears from the results of this study, as well as those based on analysis of different biochemical markers, that differentiation of leukemia cells in vitro by agents such as DMSO is reflected by major changes of enzyme and membrane protein activities.
vitro of murine [Friend] virus-induced leukemic cells. Natl Cancer
Inst Monogr 1966; 22:504-514. [7] Loritz F, BERNSTEIN A, Miller RG. Early and late volume change
during erythoid differentiation of cultured Friend leukemic cells. J
Cell Physiol 1977; 90:423-438. [8] Aviv H, Volch Z, Bastos R, LEVY S. Biosynthesis and stability of
globin mRNA in cultured erythroleukemia Friend cells. Cell 1976:
8:495-503. [9] CONKIE D, AFFARA N, Harrison PR, Paul J, JONES K. In situ
localization of globin messenger RNA formation. II. After treatment of Friend virus-transformed mouse cells with dimethyl sulf.
oxide. J Cell Biol 1974; 63:414-419. [10] Curtis PJ, Mantei N, VAN DEN Berg J, WEISSMANN C. Presence of a
putative 15$ precursor to B-globin mRNA but not to a-globin mRNA in Friend cells. Proc Natl Acad Sci USA 1977; 74:3184
3188. [11] Nudel U, SALMON J, FIBACH E, et al. Accumulation of a- and B
globin messenger RNAs in mouse erythroleukemia cells. Cell 1977;
12:463-469. [12] Ostertag W, Crozier T, Kluge N, Melderis H, DUBE S. Action of
5-bromodeoxyuridine on the induction of haemoglobin synthesis in mouse leukemia cells resistant to 5 BUDR. Nature [New Biol] 1973;
243:203-205. [13] Ross J, Ikawa Y, LEDER P. Globin messenger-RNA induction during
erythroid differentiation of cultured leukemia cells. Proc Natl Acad
Sci USA 1972; 69:3620-3623. [14] BOYER SH, Wuu KD, Noyes AN, et al. A. Hemoglobin biosynthesis
in murine virus-induced leukemic cells in vitro: Structure and
amounts of globin chains produced. Blood 1972; 40:823-835. [15] Ostertag W, MELDERIS H, STEINHEIDER G, KLUGE N, DUBE S.
Synthesis of mouse hemoglobin and globin mRNA in leukemia cell
cultures. Nature [New Biol] 1972; 239:231-234. [16] Ebert PS, Ikawa Y. Induction of S-aminolevulinic acid synthetase
during erythroid differentiation of cultured leukemia cells. Proc
Soc Exp Biol Med 1974; 146:601-604. [17] KABAT D, SHERTON CC, Evans LH, BIGLEY R, KOLER RD. Synthesis
of erythrocyte-specific proteins in cultured Friend leukemia cells.
Cell 1975; 5:331-338. [18] FURUSAWA M, IKAWA Y, SUGANO H. Development of erythrocyte
membrane-specific antigens in clonal cultured cells of Friend virus
induced tumor. Proc Jpn Acad 1971; 47:220-225. [19] MacDonalD ME, LETARTE M, BERNSTEIN A. Erythrocyte membrane
antigen expression during Friend cell differentiation: Analysis of
two non-inducible variants. J Cell Physiol 1978; 96:291-301. [20] ARNDT-Jovin DT, Ostertag W, Eisen H, Klink F, Jovin TM. Studies
of cellular differentiation by automated cell separation. Two model systems: Friend virus transformed cells and Hydra attenuata. J His
tochem Cytochem 1976; 24:332-347. [21] Eisen H, Bach R, EMERY R. Induction of spectrin in erythroleukemic
cells transformed by Friend virus. Proc Natl Acad Sci USA 1977;
74:3898-3902. [22] Sabban EL, Sabatini DD, MARCHESI VT, ADESNIK M. Biosynthesis of
erythrocyte membrane protein band 3 in DMSO-induced Friend
erythroleukemia cells. J Cell Physiol 1980; 104:261-268. [23] LORAND L. Fibrinoligase: The fibrin-stabilizing factor system of blood
plasma. Ann NY Acad Sci 1972; 202:6-58. [24] Williams-Ashman HG, Notides AC, Pabalan SS, LORAND L. Trans
amidase reactions involved in the enzymatic coagulation of semen: Isolation of y-glutamyl-e-lysine dipeptide from clotted secretion protein of guinea pig seminal vesicle. Proc Natl Acad Sci USA
1972; 69:2322-2325. [25] Anderson DR, Davis JL, CARRAWAY KL. Calcium-promoted changes
of the human erythrocyte membrane. Involvement of specirin, transglutaminase, and a membrane-bound protease. J Biol Chem
1977; 252:6617-6623. [26] SIEfRING GE Jr, APOSTOL AB, Velasco PT, Lorand L. Enzymatic
[1] Friend C, Scher W, HOLLAND JG, Sato T. Hemoglobin synthesis in
murine virus induced leukemic cells in vitro: Stimulation of erythroid differentiation by dimethyl sulfoxide. Proc Natl Acad Sci USA
1971; 68:378-382. [2] Marks PA, RIFKIND RA. Erytholeukemic differentiation. Annu Rev
Biochem 1978; 47:419-448. [3] FIBACH E, REUBEN RC, RIFKIND RA, Marks PA. Effect of hexa
methylene bisacetamide on the commitment of differentiation of
murine erythroleukemia cells. Cancer Res 1977; 37:440-444. [4] GUSELLA JF, Housman D. Induction of erythroid differentiation in
vitro by purines and purine analogues. Cell 1976; 8:263-269. [5] Preisler HD, LUTTON JD, Giladi M, Goinstein K, ZANJANI ED.
Loss of clonogenicity in agar by differentiating erythroleukemia
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and the cat as in man. When only rates for all leukemias in entire dogs and cats were compared, there was an approximate range of 20% more male expression in the dog to a 30% excess expression in the cat [table 5]. Human subpopulations in the United States also show variable percent excesses of male over female age-adjusted leukemia rates, some in the same ranges as those in the cat and the dog, but most falling between 40 and 60% or approximately two times the dog to cat excess percentages [13]. It has been hypothesized that the major reason for excess human male cancer at most sites was a more efficient immunologic system in human females [14]. Female dogs and cats also appear better able to survive in our environment than their male counterparts, for in both species, as in humans, the survival gap consistently widens between male and female with increasing age [10]. Moreover, in an unpublished analysis of mortality data for dogs and cats collected in the two population surveys used in this study, females had lower age-adjusted, disease-only mortality rates among both neutered and entire groups than their male counterparts.
In contrast to humans, dogs, and cats, other species for which there have been sufficient data available tend to have excess expression of leukemia in the female. These species are the mouse [15], rat [16], guinea pig [17], horse [7], and cow [7]. Thus the leukemia patterns of humans, dogs, and cats differ in sex expression preference from the other named species. The reasons for this difference may be important in understanding the etiology and/or pathogenesis of leukemia and why sex hormones can affect risk. The two most obvious differences are physical environment and diet. The dog and the cat tend to share closely the same physical environment of man, and some aspects of this environment may relate to male leukemia excess. It is also possible, however, that diet may be more important, in that humans, dogs, and cats eat meat and animal products as a major dietary source, whereas the other species do not. Perhaps animal-source steroid or protein metabolism is in some way involved.
In addition to male-female differences, effects of sex hormone removal via neutering were also present [text-figs. 4, 5]. The effects of neutering appeared to be related partially to species, partially to sex, and partially to the age when neutering occurred. Species and sex differences were found in the female only. In the males of both species, entire male age-adjusted rates were approximately 60% more than neutered rates, whereas for the neutered female compared to the entire female, there were marked differences between dogs and cats [tables 4, 5]. There was little or no change due to neutering in the female dog, where neutered expression was almost the same as the entire expression. However, in the female cat, neutering reduced expression by approximately 40-50% of the entire female age-adjusted rate. The reason for these female differences was not clear and should be pursued further, especially since males of both species showed the same magnitude of reduced risk from neutering. Two areas that might be fruitful for study are the differences in estrous cycles between species and the effect of pregnancy, which would generally occur more frequently in cats.
Neutering in three strains of mice [15] resulted in higher leukemia expression in those neutered, which was exactly opposite to the results shown in this paper for the dog and
the cat. Neutered male horses and cows [7] also had excess leukemia expression as was found in the mouse. Thus there appears to be a consistency of opposite neutering as well as opposite male-female effects in the dog and the cat and perhaps man also, when compared to the effects seen in rodents and herbivores.
In addition, age at neutering appears to be important in the neutered risk effect for both males and females [table 6]. It is obvious that the major part of the reduced risk effect of neutering in the feline female and male and the canine male was due to neutering before maturity [table 6]. Interestingly, there was a definite pattern of increasing and decreasing risk to the ratios obtained when prior age-adjusted, ageneutered leukemia incidence rates were divided by subsequent age-adjusted, age-neutered incidence rates [table 7]. The same pattern was present in the female regardless of species. Thus even though neutering did not affect the leukemia expression of the female dog, age at neutering showed a similar pattern of risk effect as seen in the female cat, where neutering reduced leukemia expression by approximately 40-50% [table 5]. With a slight reduction in the 4- to 8-year incidence rate of the neutered male cat, a similar ratio pattern could also be seen in males of both species, where the effect of neutering was the same [table 7]. Further work is necessary to gain an understanding of the pattern of increasing and decreasing risk effect of age at neutering.
In summary, age and sex preferences of leukemia expression in the cat and the dog resemble those in humans. The sex preference contrasts with what has been previously found in some rodent and herbivore species. In addition, neutering reduced risk markedly in the female cat but less so in the male cat and dog and did not appear to affect risk in the female dog. However, the choice of the age of neutering and the sex had a profound interacting effect on whether neutering decreased, increased, or did not change expression in both the cat and the dog.
[1] PRIESTER WA, MANTEL N. Occurrence of tumors in domestic animals.
Data from 12 United States and Canadian colleges of veterinary
medicine. J Natl Cancer Inst 1971; 47:1333-1344. [2] Priester WA. Skin tumors in domestic animals. Data from 12 United
States and Canadian colleges of veterinary medicine. J Natl Cancer
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rural areas where hygienic conditions are poor. Also, many patients reported multiple female partners. As already noted, carcinoma of the penis is prevalent in areas where there is also a high incidence of carcinoma of the cervix. Preliminary studies by Dr. Jenson suggest an association with the papillomavirus, which is not surprising in view of the epidemiologic data.
Dr. Helena de Restrepo reviewed information on lung cancer noting that in Latin America the incident rates are lower than in developed countries. As in other areas, however, lung cancer is associated with the use of cigarettes and alcohol. No differences were found, however, between the incidence of lung cancer and the different types of tobacco that are used. In Colombia, carcinomas of the larynx are more common than carcinomas of the lung. Dr. Rei T. Ravenholt indicated that cigarette smoking has caused the major health problems in the late 20th century.
Lymphomas
Dr. Gregory T. O'Conor discussed lymphomas and the need for uniformity in classification and in diagnostic criteria. He emphasized that the lymphomas and lymphoid leukemias represent a group of distinct biologic entities and should not be regarded as a single disease with morphologic variations. He noted that much of the available incidence data such as those reported in previous editions of "Cancer Incidence in Five Continents” were based on inadequate or very limited classification systems. Dr. O’Conor highlighted opportunities for epidemiologic studies of lymphoid neoplasms, since it is already evident that there are large differences in the geographic distribution of many types of lymphomas and leukemias and there are often special characteristics that may provide clues to etiology. Important examples are Burkitt's lymphoma in Africa and T-cell lymphoma in Japan. Some observations require further epidemiologic investigation. With particular reference to Latin America, they include the geographic and ethnic variations in incidence and age distribution of different types of acute lymphoblastic leukemia, the changing time trends for specific lymphomas and leukemia subtypes, the deficiency of nodular lymphomas and the excess of extranodal lymphomas in developing countries, the high frequency of nasal lymphoma in Peru, and the occurrence of adult T-cell lymphoma in the Caribbean area and its relationship to human T-cell leukemia virus. There is every indication that a better definition of lymphomas and leukemias, based on modern classification systems utilizing functional parameters as well as morphology, offers an opportunity to gain new insights into the natural history and potential control of this important group of neoplasms.
Dr. Daniel Knowles described the usefulness of cell markers for the diagnosis of lymphoproliferative cancers and emphasized those that can be measured in any pathology laboratory. He also described the antigenic changes that occur during lymphocyte differentiation.
Cell marker analysis can distinguish benign, reactive lymphoid proliferations [which are immunologically polyclonal] from neoplastic lymphoid proliferations [which are immunologically monoclonal]. In many cases, these studies can also determine the cell origin of the lymphoid cancers. Clinicopathologic correlations with therapeutic and prognostic implications, not always possible by morphologic criteria alone, have been a beneficial result from these studies.
The panel of cell markers which are most helpful consists of the immunofluorescent demonstration of la antigens and surface immunoglobulin for B-cells and sheep erythrocyte rosette formation for T-cells. This panel can divide lymphoid proliferations into 4 categories-polyclonal, monoclonal Bcell, T-cell, and non-B-, non-T-cell.
The session on cancer and nutrition was chaired by Dr. Maurice E. Shils who indicated that not only is the relation between diet and cancer complex, but also the long-term influence of diet makes research difficult. In reference to intervention programs, he concluded that there is no real evidence as to what dietary changes should be made in South America to reduce cancer.
Diets can affect the incidence of cancer in several ways. These include ingestion of carcinogens, conversion of certain substrates to carcinogens in the body, interference with the inactivation or transport of carcinogens, affecting promotion of cells already stimulated, and overnutrition. At least four substances [ß-carotene, vitamin C, selenium, and a-tocopherol] are known to ameliorate the effects of carcinogens. Despite their diverse structure, these substances have one common attribute: They are all reducing agents.
Dr. Evan A. Stein discussed the relation between serum cholesterol and cancer. Although serum cholesterol levels are related to the overall fat intake, there is an apparent inverse relationship between serum levels and mortality from cancer. Found in most epidemiologic studies, this relationship seems paradoxical in view of the association between high-fat diets and cancer.
At present, the cholesterol-cancer relationship is still controversial, since there is little evidence to support more than an association. Although most of the epidemiologic data that relate lipids to cancer have come from North America and Western Europe, little work has been done in South America, where coronary artery disease and serum lipid levels are lower.
Dr. Alfredo Lopez presented preliminary data from two case-control studies which showed that decreased vitamin A and ß-carotene serum levels correlate with an increased incidence of lung cancer in Louisiana. Vitamins E and C were also reduced in many patients. These data supported a theme prevalent at the Conference that nutritional deficiencies may not cause cancer directly, but they may increase the susceptibility to naturally occurring carcinogens. According to Dr. Lopez,' borderline deficiencies of several vitamins
may be as deleterious as a severe deficiency of only one. Since diets in the United Stated are different from those in South America, it seems that collaborative simultaneous studies on various populations could yield important information.
At the conclusion, Dr. Jorge Litvak, on behalf of The Pan American Health Organization, thanked all participants for their contribution. He emphasized that the Organization, as part of its plans to expand research on cancer epidemiology in Latin America, will assist investigators in both North and South America by arranging for contacts, preparing research proposals, encouraging investigators to submit applications to the Organization's research grant program, and assisting investigators to obtain funds from other agencies.
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In “The Causes of Cancer: Quantitative Estimates of Avoidable Risks of Cancer in The United States Today,” by Richard Doll and Richard Peto, page 1197, entry in table 1 should read: “... each contributing less than 4% of deaths” [not“. . . each contributing less than 3% of deaths”].
sium to be held on September 26-30, 1983. The main topics of the conference will be: cyclic AMP, cyclic GMP, and calcium as signals; control of intermediary metabolism by insulin; neurochemistry; steroids and gene expression; control of cell multiplication by growth factors; signals in development and differentiation.
Detailed information on the program, registration, etc., are available from Dr. J. E. Dumont, Institute of Interdisciplinary Research, Faculty of Medicine, Free University of Brussels, Campus Hôpital Erasme, 808 route de Lennik, B-1070 Brussels, Belgium.
Fourth International Congress of Dermatologic Surgery
The Journal will publish notices of major meetings dealing with cancer. Submission of notices 6 months in advance is requested to permit timely publication.
New Educational Institution—The European School of Oncology
This conference, organized by the International Society for Dermatologic Surgery and the Department of Medical and Surgical Dermatology of the Granada University, will be held in Granada, Spain, on October 25, 1983. The ISDS brings dermatosurgeons together so they may exchange innovative techniques and work for standardization and improvement of training programs in dermatologic surgery.
For registration forms and additional information please contact Dr. V. Delgado Florencio, OTECSA, Recogidas, 21-1o, Granada, Spain.
Third International Symposium on Medical Virology
The European School of Oncology, promoted by the European Society of Surgical Oncology, will present postgraduate training in the field of clinical oncology. The following is a program of courses being offered for 1983: Breast Cancer; Dr. Veronesi [Milan] Chairman; course date May 30
June 8, 1983. Principles of Medical Oncology; Dr. Pinedo [Amsterdam] Chairman;
course date June 12-19, 1983. Malignant Melanoma; Dr. Levene [London] Chairman; course date
October 24-30, 1983. Genito-urinary Tract Tumors; Dr. Denis [Brussels] Chairman; course
date November 7-13, 1983. Colo-rectal Cancer; Dr. Goligher [Leeds] Chairman; course date No
vember 21-27, 1983. All the courses will be held at the Pomerio Castle, 40 minutes from Milan. English will be the official language. Additional information and requests for participation should be addressed to the European School of Oncology, Via Venezian, 1, 20133 Milan, Italy [telephone: [02]294662].
The Medical Microbiology Division, Department of Pathology, University of California, Irvine, Medical Center will sponsor this symposium to be held at the Disneyland Hotel, Anaheim, California, on October 19-21, 1983. This is an approved program of Continuing Medical Education. Physicians attending this course may report up to 18-34 hours for formal [Category I] credit toward the California Medical Association Certificate in Continuing Medical Education and the American Medical Association Physician Recognition Award.
For further information contact Dr. Luis M. de la Maza, Department of Pathology, University of California, Irvine, Medical Center, Orange, California 92668 [telephone: 714-634-6868].
Second International Conference on Safety Evaluation and Regulation of Chemicals
Design and Analysis of Scientific Experiments Course
The Massachusetts Institute of Technology will offer this one-week course to be held on July 11-16, 1983. Applications will be made to the physical, chemical, biological, medical, engineering, and industrial sciences, and to experimentation in psychology and economics. The course will be taught by Professors Harold Freeman and Paul Berger.
Further particulars may be obtained by writing to the Director of Summer Session, Room E19-356, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
This course, sponsored by the American College of Toxicology, organized by Boston University School of Medicine, Bio-Research Institute, Cambridge, and Mallory Institute of Pathology, Boston, City Hospital, will be held at the Hotel Sonesta, Cambridge, Massachusetts, on October 2428, 1983. The first two days of this conference will focus on the safety evaluation and regulation of chemicals as viewed by representatives of state and federal governments and industry; procedures for compliance with regulations and the testing required will also be included. The last three days [International Conference] will deal with regulations of drugs, foods, cosmetics, and toxic substances throughout the world from the point of view of government and international agencies, and with method development for safety evaluation.
The Academy of Toxicological Sciences has approved accreditation for AMA Category I Continuing Medical Education credit. For additional information please contact Dr. Freddy Homburger, Bio-Research Institute, 9 Commercial Avenue, Cambridge, Massachusetts 02141 [telephone: 617864-8735].
Eighth European Symposium on Hormones and Cell Regulation
Sainte-Odile [near Strasbourg], France, will be the site of this sympo
Fifth World Congress of Cryosurgery
The Philippine International Convention Center, Manila, Philippines, will be the site of this conference to be held on November 17-19, 1983. The program will include cryobiology, cryoimmunology, cryotherapy with multi-disciplinary approach, and cryopreservation in addition to scientific sessions.
Registration rates will increase after July 31, 1983. Abstracts should be sent to Dr. Sajio Sumida, The National Fukuoka Central Hospital 2-2, Jonai, Chuoku, Fukuoka 810, Japan [telephone: 092-714-0151] for receipt no later than May 31, 1983. For additional registration information please contact Dr. Samuel M. Tanchoco, Suite 31, Lagaspi Towers, 300 Roxas Blvd. corner Vito Cruz, Manila, Philippines [telephone: 59-40-80/59-5737].
Inn, Newport, Rhode Island, on August 15-18, 1983. The symposium will focus on the use of digital imaging, NMR, computed tomography, ultrasound, nuclear medicine, angiography, and diagnostic imaging in diagnosing adult and pediatric diseases and problems of the gastrointestinal, reproductive, genitourinary, skeletal, thoracic, and neurologic systems. Invasive and noninvasive techniques and the future of the imaging modalities will be discussed in lectures and small group workshops.
Accreditation has been approved for 21-1/2 hours, Category I, AMA Physician's Recognition Award. For additional information contact Educational Resources Associates, Inc., P. O. Box 369, Brookline, Massachusetts 02146 [telephone: 617-738-8859/8861].
Safety Management Techniques Course
Fourth International Conference on the Adjuvant Therapy of Cancer
The National Safety Council will sponsor this course to be held in Dayton, Ohio, on September 12-15, 1983.
For additional information contact Mr. James C. Schaffner, Director, Dayton/Miami Valley Safety Council, 1980 Winters Bank Tower, Dayton, Ohio 45423 [telephone: 513-226-1444].
The University of Arizona Cancer Center will sponsor this conference to be held at the Tucson Convention Center, Tucson, Arizona, on March 21-24, 1984. Drs. Stephen E. Jones and Sydney E. Salmon will serve as conference co-chairmen.
The deadline for submission of abstracts [prepared in the format of AACR/ASCO] is November 1, 1983. For abstract forms or further information please contact Ms. Mary Humphrey, Conference Coordinator, University of Arizona Cancer Center, Tucson, Arizona 85724 [telephone: 602-626-6044].
Second International Congress of the T. and L. de Beaumont Bonelli Foundation for Cancer Research
“The Role of Viruses in Human Cancer" will be the subject of this meeting to be held in Naples, Italy, on September 22-24, 1983. The purpose of the conference is to provide current results ard perspectives in main areas in which viruses have been potentially associated with human malignancies. Topics will include: hepatitis B virus, herpesviruses, papovaviruses and retroviruses epidemiology, etiology, molecular biology, diagnosis, and prevention.
For additional information contact Dr. Gaetano Giraldo, V. Le Elena 17/B, 80122 Naples, Italy [telephone: [39]. [81]-685.013 - 684.047].
Second International Conference on Malignant Lymphoma
American College of Physicians Medical Oncology Review Course
Lugano, Switzerland, will be the site of this conference to be held at the Palazzo dei Congressi on June 13-16, 1984. This conference will deal with all important approaches in the diagnosis and treatment of Hodgkin's and non-Hodgkin's lymphoma in children and adults. Most lectures will be given by invited speakers. Selected controversial issues will be discussed by panels of leading experts.
The program will include free presentations and a poster session. The deadline for receipt of abstracts is January 15, 1984. For abstract forms and further information please contact Dr. F. Cavalli, Head, Division of Oncology, Ospedale San Giovanni, 6500 Bellinzona, Switzerland.
Honolulu, Hawaii, will be the site of this course to be held on October 17-22, 1983. Dr. Thomas C. Hall will be the course director.
For further information contact Ms. Maxine Topping, Postgraduate Division, American College of Physicians, 4200 Pine Street, Philadelphia, Pennsylvania 19104 [telephone: 215-243-1200 or 800-523-1546].
Fourth Annual Symposium on Diagnostic Imaging
Second International Conference on Human Tumor Markers
The Section on Radiation Medicine of Brown University School of Medicine and the Department of Diagnostic Radiology of Rhode Island Hospital will cosponsor this symposium to be held at the Sheraton-Islander
The International Society for Preventive Oncology [ISPO], the Austrian Cancer Society-Austrian Cancer League, and the Association of Clinical Scientists-USA will sponsor this conference to be held in Vienna, Austria, on February 20-22, 1984. The scientific program is designed for an update on the biology and clinical application of tumor markers. Panel discussions, poster presentations, and round table luncheons are devoted to the presentation of established and new tumor markers with emphasis on their sensitivity and specificity at various stages of tumor growth.
Page 14
The cover illustration was suggested by a special report on diet, nutrition, and cancer that appears in this issue. [See paper by Palmer and Bakshi on pages 1151-1170.] in evaluating the role of diet in carcinogenesis, a committee of the National Academy of Sciences emphasizes the importance of including fruits, vegetables, and whole grain cereal products in the daily diet. [Illustration by Ms. Betty Hebb, Medical Arts and Photography Branch, National Institutes of Health]
Text: Send one original and two copies. Text-figures [graphs, charts, and line drawings]: Send one original and two copies. Figures [photomicrographs and photographs]: Send three original glossy prints, unmounted. Tables: Send tabular material in typewritten form; do not submit tables as glossy prints to be photographed. The entire manuscript including footnotes, legends, and references must be typed double spaced. Single-spaced portions of a manuscript will be returned to the author for retyping. The mailing address is: Journal of the National Cancer Institute, Westwood Building, Room 850, 5333 Westbard Avenue, Bethesda, Maryland 20816.
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The possibility that murine lung adenomas may arise from two different cell types is an intriguing one. It assumes greater importance in connection with our continuing research, which deals with adenocarcinoma development and the Druckery effect. As Kauffman et al.' noted, papillary tumors may have malignant potential not possessed by solid tumors. Indeed, we have observed that large murine lung tumors induced by urethan appear invariably to be papillary in nature, whereas either set of morphologic characteristics may be dominant in small adenomas.
ELLEN J. O'FLAHERTY, Ph.D.
Department of Environmental Health
University of Cincinnati
Cincinnati, Ohio 45267
MICHAEL L. DOURSON, Ph.D.
Environmental Protection Agency Cincinnati, Ohio 45268
Page 16
the possibility existed that IgM antibodies could be recognized as IgG by this reagent. However, a few serum samples from carcinoma patients had the highest levels of IgG antibodies to FA. Furthermore, certain serum samples in all groups were positive for IgM antibody and not for IgG antibody [table 3]. To further substantiate these observations, we analyzed selected serum samples for IgG antibodies using a y-chain-specific enzyme-conjugated anti-human IgG reagent. Results presented in text-figure 2 reveal that the serum samples [#3 and #2] that were positive for IgG antibodies by heavy- and light-chain enzyme anti-IgG conjugate were also positive by y-chain enzyme anti-IgG conjugate. Both of these serum samples contained detectable levels of anti-FA of the IgM class. Furthermore, samples #1 and #4, which were negative by both IgG reagents, were positive for IgM antibodies. These results confirmed that certain serum samples did contain anti-FA antibodies of the IgG class and that in certain serum samples there was no association between the two classes of antibodies.
Distribution of FA Activity in Various Tumor and Normal Cells
attempted to develop ELISA to detect anti-FA antibodies in human sera and FA activity in various tumor and normal cells.
Text-figure 1 shows that anti-FA antibodies of both the IgM and IgG classes could be detected in sera from melanoma patients by the indirect ELISA method. The incidence of anti-FA antibodies in sera from cancer patients with various histologic types of cancers and normal volunteers is listed in table 3. There were no obvious differences in incidence between the sera from normal volunteers and cancer patients. The incidence of IgM antibodies was significantly higher than the incidence of IgG antibodies in sera from melanoma and carcinoma patients and in normal volunteers. These differences were statistically significant by the McNemar's test [P