A weakly tumorigenic phenotype with high MHC class-I expression is associated with high metastatic potential after surgical removal of the primary murine fibrosarcoma

We have examined the metastatic capacity of different clones of a chemically induced fibrosarcoma GR9. These clones have previously been characterized for their H-2 class-I and class-II phenotype, NK sensitivity and local tumor growth. Our present data show that clones which express low amounts of H-2 class-I antigens are poorly metastatic in a post-surgical spontaneous metastasis assay, while those expressing high levels of class-I antigens possess a high metastatic capacity. These results correlate inversely with local growth patterns of the clones. High metastatic capacity was associated with resistance to NK cells. In an experimental metastasis assay, based on intravenous administration of in vitro carried GR9 clones to syngeneic BALB/c mice, an opposite result to the post-surgical assay was obtained. Gamma-IFN treatment of B9 clones (H-2-deficient) enhanced H-2 class-I expression and diminished experimentally induced metastases. Metastatic colonies, from the spontaneous metastasis assay, obtained from different organs, showed changes in the ratio H-2K/H-2D. There was a tendency for down-regulation of the expression of H-2K molecules in H-2-positive clones and for up-regulation of H-2D expression in H-2-negative clones.     

Tumorigenicity of SEWA murine cells correlates with degree of c-myc amplification

Previous studies have shown that cells of the SEWA mouse tumor contain amplified copies of the proto-oncogene c-myc in the aberrant chromosomal structures of double minutes (DMs), homogeneously staining regions (HSRs) and C-bandless chromosomes (CMs). DMs, and to a lesser degree CMs, tend to disappear from the cells grown in vitro and again reappear after transfer back in vivo, as if DNA amplification confers a growth advantage upon the tumor cells. We have now isolated five in vitro clones that exhibit different degrees of c-myc amplification. When we inoculated cells of the different clones into compatible hosts, we found that there was a positive correlation between degree of c-myc amplification, level of c-myc RNA, and tumorigenicity. Our results lend further support to the idea that gene amplification contributes to the higher malignant phenotype, and to progression of tumors.     

Resistance to NK and metastatic potential of fibrosarcoma cells is associated with products encoded by the H-2D region.

We have used the murine 3-methylcholanthrene induced T10 fibrosarcoma tumor cell system originating in (C3II/en x C57BL/6)F1 mice (H-2b x H-2k) to elucidate the possible correlation between metastatic potential, expression of individual H-2 antigens and susceptibility to NK cells. Transfection of the non metastatic and NK sensitive IC9 cells (Db+, Kk, Kb, Kk-) with the H-2Dk gene, altered the metastatic phenotype of the parental cells, yet had no effect on the susceptibility of these tumor cells to lysis by NK and did not elicit a specific CTL response in syngeneic hosts. Variants of the metastatic and NK resistant IE7 clone (Db+, Kk-, Kb-, Kk-), lacking H-2Dk, were selected by treatment with monoclonal anti H-2Dk antibodies and complement. These variants were sensitive to NK and poorly or non metastatic. Transfection of Dk negative variants with the H-2Dk gene, resulted in the isolation of several clones which expressed a wide range of metastatic phenotypes but maintained sensitivity to NK. In addition, by cloning the cDNA of the H-2Dk gene of the metastatic T10-IE7 variant cells and analyzing its nucleotide sequence, we found four single nucleotide changes. Two of them are not expected to alter the encoded amino acids, whereas the others should result in two amino acid substitutions in the alpha-2 domain of the class I H-2Kd protein product. These changes might account, at least partially, for the failure of the transfection of H-2Dk to restore resistance to NK.(ABSTRACT TRUNCATED AT 250 WORDS)     

ras oncogene activation in human ovarian carcinoma

Samples of 37 fresh human ovarian tumor biopsies were screened to detect proto-oncogene amplification and ras mutations. Three samples showed c-K-ras2 amplification; none of the other oncogenes tested revealed any gene amplification. 5-, 25-, and 120-fold amplifications were assessed by dilution experiments and soft laser densitometry. Corresponding elevated levels of c-K-ras2 mRNA and p21 ras protein were found in the three tumors. Analysis by the polymerase chain reaction method to detect point mutations of codon 12 or codon 61 of Harvey-, Kirsten-, or N-ras showed only the wildtype sequence in all specimens. No correlation was found between ras activation and degree of tumor progression or histological subtype. DNA from one of the tumors with c-K-ras2 amplification proved to have high transforming activity in the NIH 3T3 tumorigenicity assay, but the transforming gene was not c-K-ras2.     

Amplification of protooncogenes in human ovarian carcinomas

Amplification of the proto-oncogenes c-myc, c-erbB2, c-K-ras2, c-H-ras1, c-erbA1, c-int2 and c-fms was studied by Southern-blot analysis of DNA extracted from 27 primary ovarian carcinomas. In addition, karyotype analysis and interphase cytogenetics using fluorescence in situ hybridization (FISH) were applied to control the chromosomal variability of the tumors. By these additional analyses the possibility of a false estimation of the grades of amplification should be diminished. In nearly 50% of the studied tumors a low to moderate (2- to 10-fold) amplification of one or more oncogenes was detected. The proto-oncogene amplified most frequently was c-myc (11 tumors), followed by c-erbB2 and c-Kras2 (3 tumors each), c-fms (2 tumors), and c-int2 (1 tumor). Neither an amplification of c-erbA1 nor of c-H-ras1 could be detected in any of the tumors. Some of the tumors showed a simultaneous amplification of two or three oncogenes. The pattern of proto-oncogene amplification clearly differentiates the studied ovarian cancers from breast tumors, but also differs from some previous results obtained from ovarian carcinomas.     

Generation and control of metastasis in experimental tumor systems; inhibition of experimental metastases by a tilorone analogue

The role of the chemical compound RMI 10,874DA (3,6-bis[2-(dimethylamino)-ethoxyl]-9H-xanthene-9-onedihydrochloride) in the abrogation of the metastatic spread of tumor cells was studied. Pre-treatment of BALB/c mice with the RMI 10,874DA compound (referred to below as tilorone analogue) completely eliminated lung colonization of an H-2-negative (GR9.B9) MCA-induced fibrosarcoma clone in an experimental metastasis assay. Other murine tumors, including H-2-positive and H-2-negative chemically induced fibrosarcoma clones and B16 melanoma, were also sensitive to the treatment; orally administered tilorone analogue given one day before the i.v. injection of tumor cells markedly inhibited lung colonization. The effect was not due to direct toxicity of tilorone analogue on tumor cells, but instead it was dependent on NK cells; this was suggested by the finding that anti-asialo GM₁ treatment of mice abrogated the effect of tilorone analogue. Kinetic studies of splenic NK activity in tilorone-treated mice showed a rapid boosting of NK-cell activity, the greatest stimulation occurring the day before removal of splenocytes for ⁵¹Cr-release assay against YAC-1 target cells. These kinetics correlated with the inhibition of in vivo lung colonization after tilorone analogue treatment. Inhibition of experimental tumor metastasis was dose-dependent and was observed when animals were treated the day before or the day after tumor-cell injection. Furthermore, repeated treatment of mice with this tilorone analogue significantly reduced lung colonization.     

NK sensitivity and lung clearance of MHC-class-I-deficient cells within a heterogeneous fibrosarcoma

The present study examines clonal variations in NK sensitivity in a methylcholanthrene-induced fibrosarcoma. Previous studies of clones from this tumor have shown considerable heterogeneity in H-2 expression, and an association between deleted or low levels of class-I products and increased tumorigenicity after subcutaneous implantation in immunocompetent syngeneic mice. Here, fibrosarcoma clones with no or low expression of MHC-class-I products were found to be sensitive to NK-mediated lysis, while clones with high levels of MHC-class-I expression were relatively resistant. One H-2+ (G2) and one H-2- (B9) clone were chosen for more detailed studies. Cold-target competition assays and conjugate cytotoxicity assays in agarose showed that splenic effector cells bound equally well to the H-2+ and H-2- tumor clone, although only the latter was sensitive to NK cell lysis. Treatment with 50 U/ml of rIFN-gamma for 48 hr increased the levels of H-2 expression and made both clones more resistant to NK-mediated lysis. In vivo studies with radiolabelled tumor cells showed that cells from the H-2+ clone survived better than cells from the H-2- clone in the pulmonary capillary bed after i.v. inoculation. This difference disappeared in mice treated with anti-asialo GM1 serum, known to deplete NK cell activity.     

Changes in the tumorigenic and metastatic properties of tumor cells treated with quercetin or 5-azacytidine

The effect of quercetin, a flavonoid derivative, on the transplantability (tumorigenicity) and metastatic behavior of mouse tumor cells was studied. BMT-11 c1-9 fibrosarcoma cells were treated in vitro with quercetin, and after cloning by limiting dilution, cell suspensions of each clone were injected subcutaneously (s.c.) into syngeneic C57BL/6 mice at a dose of 2 X 10(5) cells per mouse. Out of 17 clones examined, 8 were nontumorigenic in normal mice ("regressor" clones), whereas these clones were able to grow in immunosuppressed (600-rad-irradiated) mice. Furthermore, 1 out of 9 tumorigenic clones metastasized spontaneously to the lungs despite the very low metastatic potential of the parent BMT-11 c1-9 cells. In contrast, all 15 clones selected from the untreated parental line grew progressively in normal mice with no evidence of metastases. The appearance of both regressor and metastatic clones was also observed after treatment with a DNA hypomethylating agent, 5-azacytidine. These altered phenotypes resulting from treatment with both chemicals, however, were not necessarily stable if maintained in culture for several months. The data suggest that quercetin may be a useful new material for obtaining regressor or metastatic clones from parental tumor lines.     

Acquired resistance towards immune defense during metastatic progression represents a secondary phenomenon

The role of natural immune defense in the control of metastatic spread of tumor cells was evaluated by adapting an s.c.-grown, antigenic, but non-immunogenic rat fibrosarcoma (Bsp6S) to ascitic growth (BSp6A) in the strain of origin (BDX), where peritoneal cells display a high level of NK and macrophage activity. Parallel tests were performed to determine whether tumor cells can selectively adapt to non-specific immune defense, i.e. whether antigenicity of the non-immunogenic tumor remains unaltered, and whether this is accompanied by metastatic progression. During adaptation to ascitic growth the tumor line gradually lost susceptibility to NK cells and macrophages. This was due to the appearance of an increasing number of resistant clones (BSp6S, none; BSp6A, 50% of clones), which had lost binding structures for NK cells and macrophages. No alteration could be observed in antigenicity of the BSp6A variant as revealed by clonal analysis using LD of CTL. Neither BSp6S nor BSp6A cells metastasized. When the ascitic variant was retransplanted s.c. (BSp6AS), susceptibility to NK cells and macrophages was further decreased, in the sense that all clones of the BSp6AS variant became resistant. Furthermore, the BSp6AS variant had lost some of the tumor-associated antigens (TAAs) found on BSp6S and A variants. More important, upon s.c. transplantation BSp6AS regularly metastasized to the draining LN, contrary to BSp6S and BSp6A. When locally growing tumors were excised, rats implanted with BSp6AS frequently died with metastatic tumor burdens in LNs and lung, while other animals survived after excision of BSp6S or BSp6A. The data indicate a correlation between resistance to lysis, morphology and metastatic capacity. But the initial loss of susceptibility to NK cells and macrophages during adaptation to ascitic growth, which can be considered as an escape mechanism, was not accompanied by increased metastatic capacity. Hence, we suppose that with respect to metastatic progression of the BSp6AS variant, resistance to lysis by loss of binding structures represents only a secondary, but not a causative, element.     

Immunoselection by T lymphocytes generates repeated MHC class I-deficient metastatic tumor variants

Alteration of MHC class I molecule expression is a widespread mechanism used by tumor cells to evade T cell responses. It has long been proposed that the origin of these MHC class I-negative or -deficient tumor variants is T cell immune selection. However, there are no experimental or clinical data to substantiate this hypothesis, and this issue is currently the subject of debate. Here we report that an H-2 class I-negative fibrosarcoma tumor clone generated MHC class I-negative spontaneous lung metastases in immunocompetent syngeneic BALB/c mice. Interestingly, the same B9 clone generated MHC class I-positive metastatic nodes, under basal conditions, in athymic nu/nu BALB/c mice. This phenomenon was observed in the metastatic nodules generated after a period of in vivo growth but not in the primary tumors growing locally in the footpad. These findings support the hypothesis that the H-2 phenotype of metastatic nodes is influenced by the T cell repertoire of the host, since in the absence of this T cell pressure (i.e., in nude mice) the metastatic nodes 'recovered' H-2 class I expression. In addition, 2 different phenotypes were found when the metastatic nodules obtained from immunocompetent mice were treated with IFN-gamma. One phenotype, present in 83% of the colonies, was characterized by resistance of the Ld molecule to IFN-gamma induction, due to a deletion involving the Ld gene. The second phenotype (17% of the colonies) was similar to the original B9 clone and was characterized by the response of K, D and L class I genes to IFN-gamma. These data provide evidence that the changes in MHC class I expression during tumor development might not be random but could be predictable.     

Lack of correlation between DNA copy number and mRNA expression levels ofc-myc in γ-radiation-induced mouse thymic lymphomas by using quantitative real-time PCR

Background It is well documented that over-expression of thec-myc proto-oncogene occurs in the vast majority of mouse thymic lymphomas induced by γ-irradiation, evidencing the importance of this gene in T-cell lymphomagenesis. However, it remains unknown whether elevated levels ofc-myc expression are driven by extrac-myc copy numbers. Materials and methods Here we use a quantitative test on the basis of real-time PCR to determine the cellular copy number of c-myc in a set of 14 g-radiation-induced thymic lymphomas obtained from (C57BL/6J x BALB/cJ) F1 hybrid mice with increased mRNA c-myc expression. Results Since 5 out of 14 (35.7%) cases had no extra copy numbers of c-myc, gene amplification was obviously not the cause of c-myc over-expression in these tumours. In the remaining 9 tumours, c-myc over-expression was also accompanied with extra DNA copy numbers. Therefore, c-myc amplification might be a consequence of the genomic instability subsequent to the up-regulation of c-myc. However, linear regression analysis showed a lack of correlation between increasing DNA copy numbers and mRNA over expression of c-myc in these tumours (r =0.029, p=0.94). Conclusion De-regulation of c-myc does not necessarity imply amplification of this gene in these tumours. This report is, to our knowledge, the first one comparing c-myc amplification with expression in lymphomas of the T-cell lineage.     

Clonal cosegregation of tumorigenicity with overexpression of c-myc and transforming growth factor alpha genes in chemically transformed rat liver epithelial cells.

Tumorigenicity was correlated with levels of expression of the genes for transforming growth factor alpha (TGF-alpha), epidermal growth factor receptor, c-myc, c-H-ras, and c-K-ras in a series of 16 clonally derived transformed liver epithelial cell lines. The clonal lines, which varied in tumorigenicity from 0 to 97%, were established from a phenotypically heterogeneous population produced by repeated exposure of diploid WB-F344 (WB) cells to N-methyl-N'-nitro-N-nitrosoguanidine. Segregation of gene expression with tumorigenicity among clonal lines was determined by correlating rank orders of gene expression by clones relative to expression by wild-type WB cells. Only the expression of the c-myc gene correlated with tumorigenicity among all transformed clones. TGF-alpha gene expression was not correlated with tumorigenicity among all clones, but it was highly correlated with tumorigenicity among clones that expressed the c-myc gene above the median level for all clones (greater than 5-fold the level of expression by WB cells). Even high levels of expression of the TGF-alpha gene (up to 60-fold the level of expression by WB cells) were not correlated with tumorigenicity among the clones expressing the c-myc gene at levels less than 5-fold the level of expression by WB cells. Clones which simultaneously overexpressed both c-myc and TGF-alpha genes at levels above the median levels for all clones were significantly more tumorigenic than were clones which expressed either or both genes at lower than median levels. These results suggest that overexpressed c-myc and TGF-alpha genes cooperate in their association with tumorigenicity. Most of the highly tumorigenic clones that overexpressed c-myc and TGF-alpha also overexpressed the c-H-ras and/or the c-K-ras genes; clones that overexpressed neither of the c-ras genes nor the genes for c-myc and TGF-alpha were not very tumorigenic, while clones that expressed one or both c-ras genes (but not both c-myc and TGF-alpha) were variably tumorigenic over an intermediate range.     

Amplification, expression and localization of the c-myc gene in BSp73 rat tumor cell lines

Metastatic (ASML 14-1, ASmv) and non metastatic (AS17-4) cell lines of the rat BSp73 pancreatic adenocarcinoma were investigated for amplification and expression of oncogene DNA. The c-myc gene was amplified, but only in one metastatic variant, ASML. The degree of amplification (3.5-fold) increased after prolongued in vitro cultivation (17.5-fold). All three tumor cell lines expressed c-myc and ras mRNA. Ras expression was at the same level as in rat liver. C-myc expression was considerably above the level in rat liver, but also differed considerably between the metastatic variants. In the metastatic ASML cells the c-myc gene was localized by in situ hybridization on a marker chromosome derived from chromosome 7. The karyotypes of the metastatic variants are different and have no common marker chromosomes. Our results obtained with the BSp73 tumor model do not support a role of the c-myc gene in the metastatic process.     

Direct correlation between nitric oxide synthase II inducibility and metastatic ability of UV-2237 murine fibrosarcoma cells carrying mutant p53

The relationship between nitric oxide synthase II (NOS II) inducibility and the metastatic ability of UV-2237 murine fibrosarcoma cells was determined. Highly metastatic cells survived to produce numerous lung metastases after i.v. injection in syngeneic C3H/HeN mice, whereas poorly metastatic cells did not. Highly metastatic clones exhibited higher levels of NOS II than did poorly metastatic clones in response to interleukin 1alpha and IFN-gamma stimulation. Furthermore, both poorly and highly metastatic clones contained an identical p53 mutation. Overexpression of NOS II in a highly metastatic clone by transfection with NOS II gene retarded tumor growth and completely suppressed metastasis. Our data indicate that a low to moderate level of NOS II expression directly correlates with metastatic ability of UV-2237 fibrosarcoma cells carrying mutant p53 and that a high level of nitric oxide production suppresses tumor growth and metastasis.     

Inhibition of tumor growth and enhancement of metastasis after transfection of the γ-interferon gene

Cells from the spontaneous metastatic TS/A mammary adenocarcinoma of a BALB/c mouse were transfected with the murine γ-interferon (IFN-γ) gene. Six clones (IFN-γ clones) releasing between 2 and 6,000 international units (IU) of IFN-γ/ml culture medium, were compared to TS/A parental cells (TS/A-pc) and to cells transfected with neomycin resistance gene only (NEO cells). Autocrine IFN-γ up-regulated membrane expression of H-2 class-1 and Ly-6 glycoproteins, but did not alter cellular proliferation in vitro. All IFN-γ clones gave rise to progressive tumors with a growth rate significantly slower than that of tumors induced by TS/A-pc and NEO cells, and inversely correlated with the amount of IFN-γ secreted. TS/A-pc and NEO tumors displayed a marginal reactive infiltrate, whereas those formed by IFN-γy clones were massively infiltrated mostly by macrophages. In T- and NK-deficient mice the growth of tumors formed by I FN-y clones was not enhanced. In vitro tests showed that IFN-γ clone cells were markedly more lysed by macrophages than TS/A-pc and NEO cells, while they remained poorly sensitive to NK and LAK cells. These data as a whole suggest that the development of solid tumors by IFN-γ clones is primarily hampered by macrophages and not by T-lymphocytes or NK cells. When spontaneous metastatic ability was compared, 2 IFN-γ clones releasing 2-4 IFN-γ lU/ml were significantly more metastatic, while most IFN-γ clones appeared to be as metastatic as NEO cells. By contrast, following intravenous challenge, all IFN-γ clones produced 5-10 times more experimental metastases than NEO cells. The higher metastatic ability of IFN-γ clones was attributed to increased resistance to NK cells since, in NK-depleted BALB/c mice, metastatic spread of IFN-γ clones was not enhanced, whereas a 50-fold increase in the number of metastases was found upon injection of NEO cells.     

Human renal-cell carcinoma cells are able to activate natural killer cells.

We previously reported that natural killer (NK) cells that had infiltrated renal-cell carcinoma (RCC) proliferated vigorously in culture with interleukin-2 (IL-2) and lysed autologous tumor cells. In this study, we investigate the susceptibility of RCC cells to NK-cell lysis and their ability to stimulate proliferation and increase phenotypic expression and function of NK cells. Cells from primary culture of RCC (p-RCC cells) were significantly more susceptible to the lysis mediated by human NK3.3 clones than were cells from primary culture of metastatic melanomas. Both RCC-cell clones and cells from primary culture of non-tumorous kidneys were also susceptible to lysis by NK3.3 clones and IL-2-activated peripheral blood lymphocytes (PBLs). Incubation of NK3.3 clones with p-RCC cells in the absence of IL-2 induced proliferation of NK3.3 clones, whereas incubation with cells from primary culture of metastatic melanomas, K562 cells, or any others tested did not. The p-RCC cells from earlier passages were more potent inducers of NK-cell proliferation than were those from older passages. Cell-free culture supernatants of p-RCC cells with or without NK3.3 clones failed to induce NK-cell proliferation. Incubation of CD16+ NK cells purified from PBLs with p-RCC cells induced higher proliferation of the NK cells only in the presence of IL-2, whereas incubation with cells from primary culture of metastatic melanomas did not. Incubation of NK3.3 clones with p-RCC cells resulted in an increase in CD16, CD25 (IL-2 receptor-alpha), and HLA-DR antigen expression and cytotoxicity in NK3.3 clones. In summary, these results suggest that RCC cells are able to activate NK cells, potentially through cell-to-cell interaction.     

Stability of c-K-ras amplification during progression in a patient with adenocarcinoma of the ovary

We have identified a case of serous cystadenocarcinoma of the ovary in which the tumor cells display an amplification (from 10- to 20-fold) of the cellular oncogene K-ras. Normal cells purified from the malignant ascites did not show such amplification. Five consecutive samples were obtained by paracentesis over a 9-month period during which the patient received chemotherapy and underwent clinical progression. The level of c-K-ras amplification in the tumor cells did not change during this period. In studies of the tumors of 6 additional patients with adenocarcinoma of the ovary and 5 cell lines of the same histology, we have detected no other example of significant c-K-ras amplification.