Activated c-K-ras and c-N-ras oncogenes in 3-methylcholanthrene-induced BALB/c fibrosarcomas

DNAs from fourteen fibrosarcomas induced by 3-methyl-cholanthrene(MCA) in BALB/c mice were analyzed for the presence of transformingoncogenes following transfection on NIH3T3 cells. Six transfection-positiveDNAs contained an activated ras gene: four c-K-ras and two c-N-ras.These results demonstrate that c-K-ras is not the only oncogeneof the ras family activated in MCA-induced murine fibrosarcomasas was previously indicated.     

Analysis of antigenic heterogeneity within individual 3-methylcholanthrene-induced mouse sarcomas

A method of establishing sublines by trocar transplantation in vivo was used to analyze possible cellular heterogeneity with regard to tumor-specific antigens in individual methylcholanthrene-induced sarcomas. Differences in antigenic specificity were found in at least 1 of the 9 pairs of sublines obtained, respectively, from opposite poles of 9 different primary tumors. No evidence suggested such differences among 7 pairs of sublines similarly derived from later transplant generations. The lack of variation in antigenic specificity between members of subline pairs obtained from later transplant generation tumors suggested that antigenic specificity was a stable characteristic. Even when derived from later transplant generations, the sublines of a particular tumor sometimes differed in growth potential, and/or immunizing capacity, and/or responsiveness to immunity. There was little or no correlation between variations in the two parameters: immunizing capacity and responsiveness to immunity. These findings led to the interpretation that immunizing capacity and responsiveness to immunity behaved like partially independent variables, and that variations in these parameters probably depended on factors other than, or in addition to, cellular antigen content per se. The marked, random, spacial heterogeneity revealed within the tumors in cellular growth rate and antigenic properties suggests that changes seen during serial tumor transplantation are probably due to random clonal variation and subsequent selection.     

Alloantigenic phenotype of radiation-induced thymomas in the mouse

The alloantigenic phenotype of 21 radiation-induced thymomas is described. Monoclonal antibodies and conventional antisera, absorbed to remove contaminating antibodies, were used to test the thymomas directly for the presence of H-2, Ia, Ly-1, Ly-2, Ly-4, Ly-5, Ly-6, Ly-9, Ly-15, Thy-1, TL, and Qa-2 antigens, and for surface immunoglobulin. The phenotypes obtained by direct tests were also confirmed by absorption studies. The tumors were all of T-cell origin (Thy-1+Ig-) and showed a restricted heterogeneity of their cell surface phenotype, concordant with the known alloantigenic phenotypes of functional T-cells. Thus the thymomas could be classified into 7 groups based on the differing expressions of Ia, Ly-1, Ly-2, Ly-4, and Ly-6 specificities; all tumors were H-2+, Ly-5+, Ly-9+, and Ly-15+. Thus a wide variety of phenotypically diverse tumors could be detected; if these represent the clonal expansion of different functional subsets, they could provide a valuable set of functional T-cells.     

Nuclear but not mitochondrial genome involvement in 3-methylcholanthrene-induced expression of tumorigenicity in mouse somatic cells

The involvement of heritable modifications of mitochondrial DNA (mtDNA) in chemical carcinogenesis was examined by studies on the effects on tumorigenicity of interchange of mtDNA between 3-methylcholanthrene (MCA)-induced mouse tumor cells and nontumorigenic mouse cells by the cytoplast-to-cell fusion technique. The difference in propagating abilities of two types of mouse mtDNA, type 1 mtDNA of B10mtJ strain and type 2 mtDNA of C57BL/10 strain, was applied successfully for complete replacement of the host cell mtDNA by cytoplasmically transmitted mtDNA. Tumorigenicity was assayed in nude mice by inoculating 5 x 10(6) cells s.c. into the backs of the mice. The results showed that tumorigenicity was not induced in nontumorigenic cells by replacement of their mtDNA by that from MCA-induced tumor cells. Moreover, the tumorigenicity of MCA-induced tumor cells was still expressed when their mtDNA was replaced by that from normal cells with a limited life span. These observations suggest that, even if MCA treatment causes heritable modifications of mtDNA, modified mtDNA cannot induce chemical carcinogenesis and that modifications of nuclear DNA alone are sufficient for the expression of tumorigenicity.     

Nalidixic acid and oxolinic acid reversibly suppress 3-methylcholanthrene-induced cell transformation of BALB/3T3 mouse cells

The effects of nalidixic acid (Nal) and oxolinic acid (Oxl), synthetic antibacterial compounds that inhibit bacterial DNA gyrase, on 3-methylcholanthrene (MC)-induced transformation of BALB/3T3 mouse cells were investigated. Exposure of the cells to Nal or Oxl for 2 weeks at any time during 4 weeks of incubation following MC treatment suppressed MC-induced transformation. Nal and Oxl also suppressed the enhancement of transformation by 12-O-tetradecanoylphorbol-13-acetate (TPA) initiated by MC. The suppression of transformation by Nal was released by exposure of the cells to TPA after removal of Nal. Since the suppressive effects of Nal and Oxl on transformation were time-related, they may be due to epigenetic changes.     

Genetic control of susceptibility to 3-methylcholanthrene-induced subcutaneous sarcomas

Using a genetic system in which aryl hydrocarbon hydroxylase (AHH) inducibility segregates, it was observed that inducible mice are approximately 12 times more sensitive to 3-methylcholanthrene-induced tumorigenesis than their non-inducible littermates. The type of parental cross (maternal influence) plays no role in this sensitivity. Hostregulated expression of the group-specific (gs) antigen of type-C RNA viruses, although also segregating in this genetic system, does not seem to play a major role in this enhanced susceptibility to 3-methylcholanthrene carcinogenesis. Results are discussed with the view that the enhanced sensitivity of the AHH-inducible animals probably results from rapid and efficient metabolism of the chemical to its ultimate carcinogenic form.     

Characterization of spontaneous metastases from autochthonous 3-methylcholanthrene-induced tumors

In mice bearing autochthonous 3-methylcholanthrene-induced tumors metastasis was rare, with only 2 out of 47 (4%) animals showing lung secondaries and 1 showing kidney lesions. Surgical excision of autochthonous growing tumors brought only a slight increase in incidence of metastasis (5 out of 42 mice, 12%). Cell lines were established by in vivo and/or in vitro passage from two kidney metastasis found in the same host (0.13-K1 and 0.13-K3) and from a spontaneous lung metastasis found in 2 mice (mR80/43 and mR80/17) and compared to lines from the respective primary tumors (0.13; R80/17; R80/43). Cell lines from metastases and primary tumors were heterogeneous in tumorigenicity, growth rate, metastatic potential (spontaneous), and colonizing capacity (i.v. inoculation). In particular, the mR80-43 line was more metastatic to lungs upon intravenous injection than the parent R80-43 primary tumor. Similarly the 013-K1 line from a kidney secondary caused more lung nodules when inoculated intravenously than the parent 0.13 line, but this was not the case with the 013-K3 line derived from another kidney secondary in the same host. The R80-17 and mR80-17 lines had similar lung-colonizing capacity. Lung colonizing ability was not strictly correlated to the capacity to form spontaneous metastases. Changes in lung-colonizing capacity occurred in part of the lines (013, 013-K1, R80-17, mR80-17) upon in vitro or in vivo passage. These findings with lines from spontaneous metastases from three autochthonous sarcomas extend previous observations on the heterogenous behavior of transplanted metastatic neoplasms.     

Modulatory influence of oral contraceptive pills Ovral and Noracycline on 3-methylcholanthrene-induced carcinogenesis in the uterine cervix of mouse.

The present study reports the modulatory influences of combined oral contraceptive formulations, Ovral (0.05 mg ethinylestradiol plus 0.5 mg norgestrel per pill) and Noracycline (0.05 mg ethinylestradiol plus 0.1 mg lynestrenol per pill), on methylcholanthrene (MCA)-induced carcinogenesis in the uterine cervix of Swiss albino mouse. Placement of cotton thread impregnated with beeswax containing approximately 300 micrograms of MCA yielded cervical tumors in 0.0%, 8.6% and 26% animals, respectively, in 30, 60 and 90 days. Concomitant treatments with doses D1 (1/2000th of a pill), D2 (1/200th of a pill) and D3 (1/20th of a pill) of Ovral yielded cervical tumors in 0.0%, 0.0% and 4.5% mice at 30 days, 0.0%, 6.2% and 10% mice at 60 days and in 3.3% (P less than 0.05), 3.4% (P less than 0.05) and 47% mice at 90 days, respectively. Likewise, concomitant treatments with doses D1 (1/2000th of a pill), D2 (1/200th of a pill) and D3 (1/20th of a pill) of Noracycline yielded cervical tumors in 0.0%, 0.0%, 16.6% mice at 30 days, 4%, 3.7% and 54% (P less than 0.05) mice at 60 days and 3.2% (P less than 0.05), 20% and 63% (P less than 0.05) of mice at 90 days, respectively. Both Ovral and Noracycline displayed biphasic action on MCA-induced cervical carcinogenesis in mice. At lower dose levels (D1 and D2), they were inhibitory while at the higher dose level (D3) they were augmentatory in their actions. Both pills also significantly enhanced the incidence of cervical hyperplasia.     

Tumor-associated antigens in isoantigenic variants of a 3-methylcholanthrene-induced sarcoma.

A sarcoma was induced in an (A.CA X A.BY)F1 mouse. Two isoantigenic variants were selected by loss of one H-2 antigen. The tumor-associated transplantation antigens (TATA) of these variants were compared as to their specificities in (A.CA X A.BY)F1 mice. Both transplantation and indirect membrane immunfluorescence tests revealed that TATA of both variants did not cross-react. Thus selecting angainst different H-2 antigens also selected different TATA. Karyotype studies suggested that both variants originated from a unique clone.     

Genetic control of resistance to 3-methylcholanthrene-induced T-cell lymphoma in mice

Different strains of inbred mice exhibit different levels of susceptibility to T-cell lymphoma induced by the carcinogen 3-methylcholanthrene (MCA). Resistance to MCA-induced lymphoma was dominant over susceptibility in all crosses tested, and in inbred strain mice MCA resistance was found to correlate with resistance to lymphoma induced by a fractionated dose of gamma irradiation. The susceptible RF/J strain was also found to be extraordinarily sensitive to lymphoma induction by low doses of X-irradiation; this low dose sensitivity was also recessive. Experiments on both first and second generation backcross populations supported the hypothesis that a single locus is the main determinant of MCA resistance. Studies examining the possible linkage of low lymphoma incidence to a number of loci failed to establish any clear association, but linkage was seen between the Lyt-2 locus and a significant delay in MCA-induced lymphoma development. We also examined the strain distribution of several activities which might have been expected to have an effect on susceptibility to induced lymphoma. No correlation was seen between resistance and either DNA repair ability, thymic superoxide dismutase levels, or natural killer activity.     

Cell-mediated reactivity to antigens shared by Moloney-virus-induced lymphomas (LSTRA) and certain 3-methylcholanthrene-induced mouse sarcomas.

Spleen cells (SC) both from BALB/c mice whose primary Moloney sarcoma virus (MSV)-induced sarcomas had spontaneously regressed and from normal, untreated BALB/c mice, were co-cultivated for 5 days with mitomycin-C-treated LSTRA cells; LSTRA is a BALB/c Moloney lymphoma which shares cell surface antigens with MSV-indiced sarcomas. These SC, referred to as CMR and CU cells, respectively, were shown to be cytotoxic to LSTRA cells in 3 h 51Cr-release assays; CMR cells showed, in most cases, the greatest lytic activity against LSTRA targets. The same SC were also reactive, in 20-h microcytotoxicity and 51Crassays, against target cells from a variety of transplanted sarcomas indiced by 3-methylcholanthrene (MCA) in Balb/c mice. The highest reactivity was seen when CMR or CU cells were tested against target cells from sarcoma lines that expressed an NB-ecotropic MuLV cross-reacting serologically with Moloney virus. Reactivity against isotope-labelled tumor cells expressing MuLV-associated cell surface antigens could be competititively inhibited by adding unlabelled tumor cells expressing such antigens. Finally, Winn assays were performed in which CMR cells strongly inhibited the outgrowth of cells from three sarcoma lines that express the NB-ecotropic MuLV. There was less but significant inhibition of cells from some other MCA sarcomas, either negative for the expression of MuLV-associated antigens or expressing the N-ecotropic endogenous BALB/c MuLV. CU cells enhanced tumor outgrowth in Winn assays at least as often as they inhibited it.     

High frequency of ras oncogene activation in all stages of human thyroid tumorigenesis

Using polymerase chain reaction amplification and oligonucleotide probing, the activation of ras oncogenes in 24 benign and 20 malignant human thyroid neoplasms was examined. The frequency of ras oncogene activation was similar at all stages of tumorigenesis in this system, being found in 33% of adenomas overall (50% of microfollicular tumours), 53% of differentiated follicular carcinomas and 60% of undifferentiated carcinomas. This supports the contention that mutation of these oncogenes occurs at an early step in tumorigenesis. The predominant amino acid substitution in the differentiated tumours was glutamine to arginine at position 61 of Ha-ras or N-ras, but this mutation was not found in any of the undifferentiated tumours. It was noted that while transition mutations predominated in differentiated tumours (both benign and malignant), transversions were more common in the undifferentiated tumours.     

High frequency of activation of tyrosine kinase oncogenes in human papillary thyroid carcinoma

We had previously detected a transforming oncogene, designated PTC, in 25% of 20 papillary thyroid carcinomas. In order to characterize further the transforming activity of this tumour histotype, a new panel of tumour specimens from 16 patients was analysed by using a modified calcium phosphate-DNA coprecipitation transfection protocol. Tumour DNA from 10 patients (62%) displayed a transforming activity due to activation of three different oncogenes identified in four cases as PTC, in four cases as TRK, and in two cases as N-RAS. The same structural alterations of PTC and TRK (gene rearrangements) as well as of N-RAS (point mutation) detected in the NIH3T3 transformants, were also found in the original tumour DNAs, thus indicating that their activation was not due to transfection procedures. Since both PTC, a novel rearranged form of RET, and TRK display a tyrosine protein kinase activity, it is proposed that the activation of this class of oncogenes is specifically involved in the pathogenesis of papillary thyroid cancer.     

Distribution of radiolabeled alloantibodies in mice bearing 3-methylcholanthrene-induced sarcoma.

The distribution of purified 125I-labeled alloantibodies, prepared from the serum of DBA/2J mice obtained after immunization with C3H/HeJ spleen cells, was studied in immunosuppressed DBA/2J mice bearing either allogeneic C3H/HeJ 3-methylcholanthrene sarcomas growing s.c. or syngeneic SaD2 3-methylcholanthrene sarcomas. Once purified radiolabeled antibody was isolated from 125I-labeled immune gamma-globulin by a single adsorption onto C3H/HeJ RBC and elution from stroma prepared from these cells, by using 0.1 M glycine buffer (pH 3.0). Twice-purified alloantibody was then produced by Bio-Gel P-200 or Sephadex G-200 gel filtration chromatography or DEAE A-50 ion-exchange chromatography. In vitro, such purified antibodies bound specifically to C3H/HeJ RBC. In vivo, they localized to a significant extent following i.p. injection, preferentially in C3H/HeJ 3-methylcholanthrene sarcomas (4.4 to 8.9% of the injected dose per g of tumor equal to 1% of mouse weight), with mean tumor/blood ratios of 4.0 to 7.8, at 24 or 48 hr after injection. The percentage of injected dose localized in tumor and the tumor/blood ratio did not show significant differences with respect to time or method of antibody purification. Normal tissue/blood ratios in C3H/HeJ or SaD2 sarcoma-bearing mice were less than 0.9. The tumor/blood ratios in SaD2 sarcomas were approximately 0.6. Injection of 131I-labeled normal DBA/2J gamma-globulin resulted in normal tissue/blood and tumor/blood ratios of less than 0.9 in C3H/HeJ tumor-bearing mice.     

High stereoselectivity in mouse skin metabolic activation of methylchrysenes to tumorigenic dihydrodiols

The stereoselectivity of mouse skin metabolic activation to dihydrodiols of the strong carcinogen 5-methylchrysene (5-MeC) and the weak carcinogen 6-methylchrysene (6-MeC) was investigated. Synthetic 1,2-dihydro-1,2-dihydroxy-5-methylchrysene (5-MeC-1,2-diol), 5-MeC-7,8-diol, and 6-MeC-1,2-diol were resolved into their R,R- and S,S-enantiomers by chiral stationary phase high performance liquid chromatography. The absolute configurations of the enantiomers were assigned by their circular dichroism spectra. Using these enantiomers as standards, the metabolism of 5-MeC and 6-MeC in vitro in rat and mouse liver and in vivo in mouse epidermis was investigated. Only the R,R-enantiomers of each dihydrodiol predominated (greater than 90%). The dihydrodiol enantiomers were tested for tumor initiating activity on mouse skin. In each case, the R,R-dihydrodiol enantiomer was significantly more tumorigenic than the S,S-enantiomer. The most tumorigenic compound was 5-MeC-1R,2R-diol; it was significantly more active than either 5-MeC-7R,8R-diol or 6-MeC-1R,2R-diol. The results of this study demonstrate that there is a high degree of stereoselectivity in the metabolic activation of 5-MeC and 6-MeC to proximate tumorigenic dihydrodiols in mouse skin. The bay region methyl group has no effect on the stereoselectivity of activation to 1,2-dihydrodiol metabolites in the chrysene system.     

3-methylcholanthrene-induced monooxygenase (O-deethylation) activity of human lymphocytes.

With a direct fluorescence assay, the levels of mixed-function oxidase activity were determined in mitogen-activated human lymphocytes. The O-deethylation of ethoxyresorufin to resorufin was used to quantitate mixed-function oxidase activity. Ethoxyresorufin O-deethylase activity was low to nondetectable in noninduced, mitogen-activated cells, but it was readily detected in 3-methylcholanthrene, mitogen-activated lymphocytes. The activity was: (a) dependent on assay time and number of lymphocytes; (b) dependent on the presence of reduced nicotinamide adenine dinucleotide phosphate; (c) stable to freezing at -80 degrees for at least 2 weeks; (d) reproducibly detected in duplicate samples of blood from one individual when cultured and assayed at the same time; but (e) quite variable in samples of blood from one individual at different times. Since in hepatic and pulmonary tissue of model animal systems ethoxyresorufin is a specific substrate for cytochrome P-448-associated monooxygenases, the use of this chemical could proffer an assay that specifically measures human cytochrome P-448-associated activity.     

Direct sequencing analysis of exon 1 of the c-K-ras gene shows a low frequency of mutations in human pancreatic adenocarcinomas

We have applied direct dideoxy sequencing of DNA fragments amplified in vitro by the polymerase chain reaction to the detection of mutations in exon 1 of the c-K-ras gene in human pancreatic adenocarcinomas. Four fresh frozen primary tumors, one metastatic tumor, and twelve formalin-fixed paraffin embedded tumors were analysed. Only three cases showed a possible mutation in codon 12 in a small population of cells, in contrast to the high frequency reported for this alteration with the RNAase A protection assay and allele-specific oligoxynucleotide hybridization. No major difference in sensitivity was found between DNA sequence analysis, and the latter method. Our results suggest that if c-K-ras mutations are indeed present in pancreatic adenocarcinomas at the high frequency reported by others, they must be confined to a small fraction of the cell population to escape detection by direct sequencing. Such a phenomenon would have implications for c-K-ras mutations in the pathogenesis of pancreatic adenocarcinomas.