Translocations among antibody genes in human cancer.

The characteristic chromosomal translocations that occur in certain human malignancies offer opportunities to understand how two gene systems can affect one another when they are accidentally juxtaposed. In the case of Burkitt lymphoma, such a translocation joins the cellular oncogene, c-myc, to a region encoding one of the immunoglobulin genes. In at least one example, the coding sequence of the rearranged c-myc gene is identical to that of the normal gene, implying that the gene must be quantitatively, rather than qualitatively, altered in its expression if it is to play a role in transformation. One might expect to find the rearranged c-myc gene in a configuration that would allow it to take advantage of one of the known immunoglobulin promoters or enhancer elements. However, the rearranged c-myc gene is often placed so that it can utilize neither of these structures. Since the level of c-myc messenger RNA is often elevated in Burkitt cells, the translocation may lead to a deregulation of the c-myc gene. Further, since the normal allele in a Burkitt cell is often transcriptionally silent in the presence of a rearranged allele, a model for c-myc regulation is suggested that involves a trans-acting negative control element that might use as its target a highly conserved portion of the c-myc gene encoding two discrete transcriptional promoters.

Abelson murine leukemia virus-induced thymic lymphomas: transformation of a primitive lymphoid precursor.

For the investigation of whether Abelson murine leukemia virus (A-MuLV) is able to transform in vivo lymphocytes other than those of the B-cell lineage, newborn BALB/c and C57BL/6 mice were given an injection of A-MuLV directly into the thymus. Thymic lymphomas appeared with a short latent period of 4-5 weeks in BALB/c mice and 8 weeks in C57BL/6 mice. Cell lines derived from some thymic lymphomas presented a very immature phenotype and did not express cellular markers of either T-cells (Thy 1.2, Lyt 1.2, and Lyt 2.2) or B-cells (cytoplasmic IgM) even after treatment with several differentiation inducers. Molecular analysis showed that T-cell receptor (TCR) beta chain genes were never rearranged; in one case only, rearrangement of TCR gamma chain genes could be demonstrated, confirming the immaturity of the presumptive T-cell lines studied. Furthermore, the cell lines consistently carried diversity (D)-joining (J) but not variable (V)-D-J rearrangements of the immunoglobulin heavy chain genes. On the whole, these findings suggest that following intrathymic A-MuLV injection neoplastic transformation does involve lymphocytes possibly of T-cell lineage, at a very early stage of differentiation.

Dependence of asbestos- and mineral dust-induced transformation of mammalian cells in culture on fiber dimension.

The abilities of chrysotile and crocidolite asbestos, glass fibers of differing dimensions, and nonfibrous mineral particulates to induce morphological transformation of Syrian hamster embryo cells in culture were compared. Chrysotile and crocidolite asbestos induced morphologically transformed colonies which were indistinguishable from transformed colonies observed following treatment with known chemical carcinogens. A linear, dose-dependent increase in the frequency of transformed colonies was observed. The slope of the dose-response curve on a log-log scale was approximately 1, which is consistent with a one-hit mechanism for their induction. The transforming potency of chrysotile asbestos was decreased by milling of the fibers but not by extraction with an organic solvent. Chrysotile asbestos was nearly twice as potent in inducing morphological transformation as crocidolite asbestos. Glass fibers were also very active in this assay. Thin glass fibers with an average diameter of 0.1 to 0.2 micrometer were as active as asbestos. In contrast, two nonfibrous particulates, alpha-quartz and Min-U-Sil, were inactive over the same concentration range used for the fibrous dusts; however, both were active at higher doses. The effect of varying fiber dimension on induction of morphological transformation was examined with glass fibers. When compared on a per-weight basis, thick glass fibers [average diameter, 0.8 plus/minus 0.06 micrometer (S.E.)] were 20-fold less potent than thin fibers [average diameter, 0.13 plus/minus 0.005 micrometer] in inducing cell transformation. When the average fiber length of thin glass fibers was reduced from 9.5 to 1.7 micrometer by milling the fibers in a mortar and pestle, a 10-fold decrease in transforming activity resulted. When the average fiber length was reduced to 0.95 micrometer, transforming ability was totally absent. The cytotoxic potencies of the various mineral dusts correlated with their transforming potencies. The varying abilities of the mineral dusts to induce cell transformation in vitro are similar to their abilities to induce mesotheliomas in vivo. Thus, this system provides a unique model for studying the mechanism of mineral fiber tumorigenesis and for comparing the relative risks of mineral dusts.

Oncogene-mediated multistep transformation of C3H10T1/2 cells.

We have examined the response of the mouse embryonic cell line C3H10T1/2 to transfection with the activated human c-H-ras oncogene and the gag-myc oncogene from avian myelocytomatosis virus 29. C3H10T1/2 cells are not morphologically transformed following transfection with the gag-myc oncogene. A low level of focus formation is observed following transfection of the c-H-ras oncogene. When C3H10T1/2 cells are cotransfected with the ras and myc oncogenes, focus formation is increased by an average of 13 fold. In addition, C3H10T1/2 ras/myc foci have a distinct, transformed morphology which correlates with an increased potential for anchorage-independent growth. Although morphological transformation in this system is largely a function of ras oncogene expression, our studies demonstrate that it is potentiated by the presence of a functional gag-myc protein. Oncogene-mediated multistep transformation, which was first described in primary embryo cultures, is not a general property of established cell lines. The C3H10T1/2 cell line is an exception and provides a model system in which partially transformed phenotypes, in a progression toward malignant transformation, can be isolated and studied.

Growth in culture and tumorigenicity after transfection with the ras oncogene of liver epithelial cells from carcinogen-treated rats.

Two epithelial cell lines designated LE/2 and LE/6 were established from cells isolated by centrifugal elutriation from the livers of carcinogen-treated rats. Both cell lines exhibit some characteristics of fetal liver cells, such as the expression of the 2.3-kilobase alpha-fetoprotein mRNA, aldolase A, and lactate dehydrogenases 4 and 5. Primary cultures contain gamma-glutamyl transferase-positive cells which do not proliferate in vitro. After the first passage, the LE/2 and LE/6 cell lines are uniformly gamma-glutamyl transferase negative. Neither cell line is transformed as assayed by morphology, anchorage-independent growth, or tumor formation in nude mice. By the 50th passage, LE/6 cells form numerous colonies in soft agar in the presence of epidermal growth factor, while no colonies grow in medium lacking this growth factor. Clonal cell populations derived from five epidermal growth factor-induced soft agar colonies were not tumorigenic in nude mice. This indicates that, although epidermal growth factor-responsive late passage cells had acquired some of the phenotypic properties commonly associated with tumor cells, these cells were not fully transformed. Transformation of LE/6 cells was accomplished by transfection of the rasH oncogene (EJ). Subcutaneous inoculation of rasH (EJ)-transfected LE/6 cells produced tumors at the site of injection with histological features of moderate to well-differentiated trabecular hepatocellular carcinomas. Tumor cell lines derived from the nude mouse tumors are gamma-glutamyl transferase positive and express alpha-fetoprotein mRNA. One clonal cell line expresses both alpha-fetoprotein and albumin mRNA. These results show that nonparenchymal liver epithelial cells transfected with an activated oncogene can give rise to differentiated hepatocellular tumors similar to those induced in livers of rats fed a carcinogenic diet.

A threshold effect in the induction of tumorigenicity of an established human cell line by v-mos.

The nontumorigenic immortal human cell line, SV80, was transfected with the v-mos gene to assess the gene's effect on tumorigenicity of cultured human cells. Two classes of cells, each containing functional v-mos, were obtained. The first class contained low levels of v-mos RNA, was morphologically transformed, but was nontumorigenic in nude mice. The second was also morphologically transformed, but contained high levels of v-mos RNA and was tumorigenic. The results indicate that SV80 cells behave similarly to murine fibroblasts in their response to v-mos in that they can be rendered tumorigenic by the viral oncogene. However, tumorigenicity was effected through a mechanism which involves different threshold doses for morphologic and tumorigenic transformation.

Transforming DNA sequences of human hepatocellular carcinomas, their distribution and relationship with hepatitis B virus sequence in human hepatomas.

Several related human transforming DNA sequences, hhc, and a putative normal liver homologue, c-hhc, have been molecularly cloned from the genomic DNAs of individual African and Asian hepatomas and from normal liver respectively. hhcM (Mahlavu) and hhcK3 (Korean), but not c-hhc, transformed NIH3T3 cells in DNA-mediated gene transfer assays. Transformed cells were found tumorigenic in athymic NIH Swiss nu/nu mice. In view of recent epidemiological studies implicating hepatitis B virus (HBV) infection early in life as causative for the eventual development of primary hepatocellular carcinoma in humans in Southeast Asia, the Far-East, and certain areas of Africa, we hereby analyzed the relationship between these hhcs and HBV in a survey of 20 hepatomas for DNA sequences homologous to hhcM and HBV by sequential hybridizations against [32p]hhcM and [32p]HBV probes. hhcM related DNA sequence were found highly amplified in 80% of the 20 hepatomas but HBV DNA sequence was rare or low. hhcM lends itself as a marker for human hepatomas. However, overall results indicated that patients with integrated HBV DNA sequences showed high copy number of hhcM sequence. Furthermore, EcoR1-restricted hepatoma DNAs showed that HBV and hhcM DNA sequences resided at different fragments in hepatomas. Our results suggest that HBV contributes to hepatocarcinogenesis probably via an activation mechanism involving possibly an integration or transient interaction of HBV DNA with hepatocyte DNA sequences, leading to recombination and eventual amplifications of the hhcM sequence in Mahlavu.

Evidence for a multistep pathogenesis in the generation of tumorigenic cell lines from hemopoietic colonies exposed to Abelson virus in vitro.

The present studies were undertaken to investigate the ability of Abelson murine leukemia virus (A-MuLV) to transform cells derived in vitro from pluripotent hemopoietic progenitor cells of high proliferative potential. We now report that continuously growing, autonomous cell lines could be obtained from a high proportion of individually infected multilineage colonies generated in assays of spleen cells from normal adult mice if the infected cells were cocultivated for the first two to three months with irradiated NIH-3T3 cells. No lines were obtained if the 3T3 cell feeders were not initially present. Similar results were obtained when the cells exposed to virus were from multilineage colonies originating from isolated single cells obtained by replating small blast colonies. Characterization of the transformants and a number of derivative cloned sublines revealed the consistent presence of a mast cell phenotype, with some suggestion of macrophage differentiation in a few cases. All cell lines tested produced virus, showed a variable pattern of A-MuLV integration, and gave rise directly to tumors when injected subcutaneously, as shown by both Southern analysis and cytogenetic studies. The early absolute but transient dependence of these A-MuLV mast cell transformants on a fibroblast feeder suggests a multistep process in their evolution, in which the acquisition of autonomy from factors of mesenchymal cell origin may play an important role.

Characterization of the transformation properties of Shope fibroma virus.

To investigate if Shope fibroma virus (SFV), a leporipoxvirus that induces benign tumors in adult rabbits, can trigger the second step of carcinogenesis in vitro or malignant transformation, an already immortalized rabbit cell line (SIRC) was inoculated with ultraviolet-irradiated virus. The resulting cell transformants displayed the characteristic properties of the malignant phenotype: lack of infectious particles, low serum requirement, high efficiency of cloning, resistance to superinfection, presence of viral DNA sequences in the nucleus, expression of viral proteins and induction of tumors in rabbits. However, this transformation was not stable since in all cell lines studied, a loss of the malignant phenotype was recorded close to the 50th passage. To assess the oncogenic potential of SFV, NIH 3T3 cells were transfected with SFV DNA. The results of these experiments indicate that SFV DNA can induce the formation of foci in certain NIH 3T3 cell lines. Taken together these results support the notion that SFV can elicit the transformation of cells in vitro.

Thyroid hormone affects the expression of neoplastic transformation induced by DNA-transfection.

Thyroid hormone can dramatically modulate oncogenic transformation of cells in culture. To further investigate this we have used DNA-mediated gene transfer (transfection) to transform cells grown in the presence (+T3) or absence (-T3) of thyroid hormones. Removal of thyroid hormones from the culture media greatly reduced the appearance of transformed foci subsequent to transfection. However, +T3 or -T3 media had no effect on the appearance of ouabain-resistant (ouar) colonies following transfection of ouabain-sensitive (ouas) cells with DNA isolated from ouar cells and selection in 3 mM ouabain. These results suggest that thyroid hormone does not effect the uptake or integration of exogenous DNA, but instead may modify the expression of transformation.

Carcinogen risk assessment.

A molecular biological rationale for the linear nonthreshold dose-response pattern for carcinogenesis is presented based on the mutagenic activation of oncogens as the basis of initiation. The approach assumes that the linear nonthreshold dose pattern at very low doses applies only to tissues that are promoted by intrinsic and extrinsic agents other than the one being modeled, and that risk is characterized on a relative rather than absolute basis in terms of aggregate tumor response.

Multiple mechanisms for the carcinogenic effects of asbestos and other mineral fibers.

Asbestos and other mineral fibers are carcinogenic to humans and animals but differ from many carcinogens in that they do not induce gene mutations. An understanding of these interesting human carcinogens, therefore, is an important problem in cancer research. Asbestos and other fibers induce predominantly two types of cancers: mesotheliomas and bronchogenic carcinomas. Fiber size is an important factor in the carcinogenic activity of these substances as has been shown for mesothelioma induction. For bronchogenic carcinomas, but not for mesotheliomas, a synergistic effect of asbestos exposure and cigarette smoke has been observed in humans. The mechanisms by which fibers alone versus fibers in concert with other carcinogens induce cancers are probably distinct. In addition to fiber dimensions, fiber durability and surface properties of fibers are important properties affecting carcinogenicity. Evidence exists that asbestos is a complete carcinogen, an initiator and a promoter. Multiple mechanisms must be operative to explain the diverse effects of mineral fibers. Although asbestos is inactive as a gene mutagen, there is now clear evidence that it induces chromosomal mutations (aneuploidy and aberrations) in a wide variety of mammalian cells including mesothelial cells. Asbestos also induces transformation of cells in culture including mesothelial cells and fibroblasts. A mechanism for cell transformation, which is dependent on fiber dimension, has been proposed. The fibers are phagocytized by the cells and accumulate in the perinuclear region of the cells. When the cell undergoes mitosis, the physical presence of the fibers interferes with chromosome segregation and results in anaphase abnormalities. The transformed cells show aneuploidy and other chromosome abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficient malignant transformation of rat embryo fibroblasts by genomic DNA from Walker carcinoma cells.

DNA isolated from Walker carcinoma ascites cells was transfected into primary rat embryo fibroblasts (REF), selecting transformed cells by growth in soft agar after prolonged propagation in monolayer. Both high molecular weight genomic DNA and a partially purified mitochondrial DNA fraction were able to transform REF with high efficiency, whereas pure mitochondrial DNA failed to elicit a transformed phenotype. Hybridization experiments showed that the mitochondrial DNA fraction contained DNA species of presumably extramitochondrial origin. Colonies were cloned into morphologically transformed, foci-forming, immortalized cell lines, showing different degrees of chromosomal alterations, tumorigenicity, and production of cell growth factors. These results indicate that although REF are refractory to genomic neoplastic DNA or to single cloned oncogenes in the absence of enhancers, they can be efficiently transformed by chromosomal DNA from a highly malignant tumor under conditions selecting against the remaining normal cells.

Continuous-wave ultrasound and neoplastic transformation in vitro.

C3H/10T1/2 cells in suspension were assayed using an initiation-promotion protocol for neoplastic transformation induced by continuous-wave ultrasound. Cells were insonated at 1.765 MHz for 40 min. Two ultrasonic intensities were used: 1.3 and 2.6 W/cm2 spatial average. The first intensity was found to be noncytotoxic; the second resulted in immediate lysis of 20% of the cells, followed by the clonogenic survival of 64% of the remaining cells. Ultrasound was delivered alone or in combination with X-rays (2 Gy, 240 kVp given before ultrasound), and/or 12-O-tetradecanoyl-phorbol-13-acetate (TPA, 0.1 microgram/ml post-irradiation). Under all treatment conditions, there was no effect of ultrasound on transformation at the 95% confidence level.

A theory of carcinogenesis based on an analysis of the effects of carcinogens.

Carcinogenic stimuli appear to act on target cells (and their daughters) by one or more of three mechanisms. The first is by oxidation of membrane component molecules on the extracellular surfaces of their plasma membranes. The second is by chronic and continuous impingement of electrons on the extracellular surfaces of their plasma membranes and the third is by relocation of predominantly basic molecules to the cytoplasmic surfaces of their plasma membranes. This latter effect in turn causes electrostatic attraction of image charged acidic molecules to the extracellular surfaces to balance the transmembrane charge of the target cells. Each of the above mechanisms results in a condition of increased electronegativity of the extracellular surfaces of plasma membranes of the target cells and their daughters. A theory of transformation is advanced based on the above related modes of action and it is used to explain some previously unexplainable properties of tumors.