The ras oncogenes increase the intrinsic resistance of NIH 3T3 cells to ionizing radiation.

Identification of genes that function to protect cells from radiation damage is an essential step in understanding the molecular mechanisms by which mammalian cells cope with ionizing radiation. The intrinsic radiation resistance (D0) of NIH 3T3 cells was markedly and significantly increased by transformation with ras oncogenes activated by missense mutations. This radiobiologic activity appeared to be a specific consequence of the ras mutations rather than of transformation, since revertant cells that contained functional ras genes (but were no longer phenotypically transformed) retained their increased D0's.

Rapid viral induction of plasmacytomas in pristane-primed BALB-c mice.

Strain BALB/c mice were injected intraperitoneally with 0.5 milliliter of pristane, and 39 to 56 days later they were infected with Abelson murine leukemia virus, which is a lymphosarcomagenic variant of Moloney virus. Fifty-eight percent of the mice developed lymphosarcoma, and 28 percent developed immunoglobulin-producing plasmacytomas within 20 to 93 days (77 to 149 days after the pristane injection). Two of 57 control mice developed plasmacytomas at days 138 and 166 after a single injection of pristane; no plasmacytomas were found in mice treated with virus alone.

Depletion of c-myc with specific antisense sequences reverses the transformed phenotype in ras oncogene-transformed NIH 3T3 cells.

ras oncogene-transformed NIH 3T3 cells expressing glucocorticoid-inducible antisense c-myc cDNA transcripts at levels sufficient to deplete c-myc protein lost their transformed morphology and the ability to grow in soft agar; their ability to form tumors in nude mice was also impaired. These changes were dependent on the continuous expression of the antisense sequences. No major effects on plating efficiencies, growth rates in monolayer culture, or immortalization were observed in the revertant cells, indicating that the observed effects were not a toxic consequence of c-myc protein depletion. Transfection with the same vector expressing c-myc in the sense orientation or other control vectors had no effect on transformation. These results suggest that a certain minimum level of expression of c-myc is required for the maintenance of ras transformation in NIH 3T3 cells.

Increased resistance to cis-diamminedichloroplatinum(II) in NIH 3T3 cells transformed by ras oncogenes.

The genetic basis of cellular resistance to the anticancer drug cis-diamminedichloroplatinum(II) (CP) is not well understood. In the course of identifying genes from human tumors capable of conferring resistance to CP, we tested the ability of several types of cellular and viral ras oncogene (H, K, and N) to alter the CP response of mouse cells. Using clonogenic assays, we found that NIH 3T3 fibroblasts transformed with missense mutation-activated ras oncogenes demonstrated substantially increased resistance to 1-h exposures to CP (P less than 0.05 to less than 0.001, at different drug concentrations), with 50% inhibitory concentration ratios (compared to NIH 3T3) of 4.5-8.5. Cells transformed with v-mos v-fms, and with a normal ras protooncogene activated by overproduction driven by an MLV ltr, demonstrate intermediate resistance (50% inhibitory concentration ratio, approximately 2.0). Cells transfected with the pSV2neo plasmid or with human genomic DNA that is not transforming had survival curves no different from those of NIH 3T3. ras genes are highly conserved in mammalian cells. Should these findings also prove to apply to human tumors, the presence of activated ras genes might help predict clinical response to CP.

Modulation of cis-platinum resistance in Friend erythroleukemia cells by c-myc.

The molecular genetic basis for cancer cell resistance to the important family of platinum coordination complex drugs is poorly understood. The observation that malignant cells commonly become resistant to therapy, while normal cells rarely do, suggests that certain molecular processes involved in malignancy, e.g., oncogene activation, might also play a role in drug resistance. Since aberrant expression and amplification of various myc oncogenes have been implicated in the prognosis of human cancers, the poor therapeutic response of some of these tumors might be explained if high levels of myc gene products rendered cells more refractory to therapy. We tested this hypothesis by determining the effect of varying the level of c-myc expression on resistance to cis-platinum and ionizing radiation in Friend murine erythroleukemia cells expressing varying levels of c-myc gene product. We found that a) the degree of cis-platinum resistance correlated directly with the level of c-myc expression, b) glucocorticoid induction of murine mammary tumor virus promoter-driven c-myc sequences significantly increased cis-platinum resistance, c) restoring c-myc transcript levels to normal restored the original cis-platinum sensitivity at a rate which paralleled that of induced c-myc transcript depletion, and d) c-myc transcript level had no effect on ionizing radiation response. These findings suggest that c-myc levels may influence therapeutic success in some tumors and may regulate specific processes by which cells cope with DNA damage caused by DNA cross-linking agents such as the platinum analogues, but not ionizing radiation.

Isolation of activated ras transforming genes from two patients with Hodgkin's disease.

Despite impressive advances in the clinical management of Hodgkin's disease, little is known about its cellular origin or the mechanism(s) of "Hodgkinogenesis." Recent findings that certain human cellular oncogenes can cause malignant transformation suggest that aberrant activation of these genes may play a role in carcinogenesis. To determine if such genes are operative in Hodgkin's cells, we isolated DNA from splenic nodules of three patients with nodular sclerosis Hodgkin's disease and tested its ability to transform mouse NIH 3T3 cells, the standard assay for oncogene-mediated malignant transformation. Transformed cells containing human DNA were obtained from two patients. DNA from these primary transformants yielded secondary transformants of NIH 3T3 fibroblasts; one also transformed normal mouse bone marrow macrophages, a cell type probably related to the Hodgkin's cell. When analyzed by Southern blot methods for homology with closed oncogene probes, the transforming genes from both patients had homology with N-ras. The homology and size of the restriction fragments were similar to those of transforming genes isolated from patients with acute nonlymphocytic leukemias. The presence of the same activated oncogene in tumor tissue from two different patients suggests that it may play an important role in Hodgkinogenesis.

Initiation of oncogenic transformation of mouse lymphocytes in vitro by Abelson leukemia virus.

While C-type RNA viruses are known to induce leukemias and lymphomas, oncogenic transformation of lymphoid cells by them in vitro has not been reported. In this study, splenocytes from female BALB/c mice were infected in vitro with Abelson virus, a C-type RNA virus that induces nonthymic lymphomas and plasmacytomas in mice. The cells were transplanted into recipients of different karyotype, either male BALB/c mice or hybrid BALB/c x AL/N (CALF1) mice, which bear the Rb(5.19)1Wh translocation. Transplants of eight of the resulting tumors (one plasmacytoma and seven lymphomas) contained cells of donor BALB/c karyotype, indicating that transformation of splenocytes occurred in vitro.

Ribosomal alterations controlling alkaline phosphatase isozymes in Escherichia coli.

Different patterns of isozymes were obtained by starch-gel electrophoresis of alkaline phosphatase from Escherichia coli strains differing only by strA or ram mutations, or both, in the 30S ribosomal subunit. The isozyme spread was reduced in strA and increased in ram strains; this strictly parallels the restriction and enhancement of translational ambiguity produced by these mutations. Streptomycin present during growth had an effect similar to ram on both isozymes and ambiguity. The three isozymes analyzed have different N-terminal residues: aspartic acid, valine, and threonine. Different patterns of isozymes were also obtained in a wild-type strain through the specific action of exogenous arginine. A link between the mechanism of the effect of arginine and that of the ribosome is not obvious. The possibility is discussed that in both cases, although by different mechanisms, N-terminals are formed with different sensitivity to limited degradative attack.

Transformation of mouse bone marrow cells by transfection with a human oncogene related to c-myc is associated with the endogenous production of macrophage colony stimulating factor 1.

We recently derived a series of transformed cell lines by transfecting mouse bone marrow cells highly enriched for macrophage progenitors with a newly described human gene, R-myc, which has homology to the c-myc oncogene. In this report, we show that these lines share some features characteristic of cells of the mononuclear phagocyte lineage. Specifically, all cell lines had macrophage- or monocytelike morphology, contained nonspecific esterase, were phagocytic for latex beads, secreted lysozyme, bore the Mac-1 antigen, and contained a minority of cells with Fc receptors. However, only a single monocytelike clone had appreciable numbers of cells which bore complement receptor 1, and none were phagocytic for antibody or complement-coated particles, or constitutively secreted Interleukin-1. All these cell lines secreted a growth factor capable of supporting the in vitro proliferation of bone marrow macrophages. Radioimmunoassay and receptor binding studies indicate that this factor is colony stimulating factor 1.

Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines.

Three Abelson virus-transformed lymphoma cell lines were established in tissue culture and the immunoglobulin biosynthesis by these cell lines was studied. Two of the cell lines (ABLS-1 and ABLS-5) were found to synthesize monomeric IgM molecules which were deposited in the cell membrane, probably to serve as an antigen receptor. The third cell line (ABLS-8) was found to synthesize membrane-associated IgM as well as cellular IgG molecules. In addition, these cell lines were found to synthesize a protein of 35,000 molecular weight which is also membrane-associated and which has the capability to bind the immunoglobulin (MAID). It is speculated that this protein might play a role in adapting the receptor immunoglobulin molecule to the hydrophobic environment of the cell membrane. The kinetics of amino acid incorporation into immunoglobulins by these cell lines show that they produce immunoglobulins at a rate which is two orders of magnitude smaller than plasmacytoma cells (MOPC 104E). These results suggest that Abelson virus transforms thymus-independent lymphocytes in various stages of maturation and these lymphocytes might be of B cell origin. The T lymphoma (P1798) used as a control cell line was found occasionally to produce minute amounts of immunoglobulin.

Increased cell-substrate adhesion accompanies conditional reversion to the normal phenotype in ras-oncogene-transformed NIH-3T3 cells.

We recently reported (1991, Mol. Cell Biol. 11, 3699-3710) that depletion of c-myc protein by myc antisense sequences in ras-transformed NIH-3T3 cells reverses several of the malignant characteristics of these cells. These include transformed morphology, growth in soft agar, and ability to form tumors in athymic mice. In the present study we examined the same cells for in vitro adhesive behavior. Cells depleted of c-myc protein by antisense transfection showed no change in attachment to fibronectin-coated dishes as compared to ras-transformed NIH-3T3 cells but had greatly increased resistance to trypsin/EDTA-mediated release from the substratum after attachment. In concomitant studies, the cells were examined for fibronectin biosynthesis and cell surface fibronectin. There was no overall change in fibronectin biosynthesis in the c-myc antisense transfected cells as compared to the ras-transformed NIH-3T3. However, immunofluorescence staining revealed increased amount of surface fibronectin associated with the antisense c-myc-expressing transfectants. Taken together, these data indicate that the conditional reacquisition of the nonmalignant phenotype in ras-transformed NIH-3T3 cells by selected depletion of c-myc protein is associated with an increase in cell-substrate adhesion. This, in turn, is associated with an increase in surface fibronectin.