Differences in the responsiveness of splenic, lymph node, and peripheral blood lymphoid cells to tumor membrane extracts.

The blastogenic response of normal spleen, lymph node, and peripheral blood lymphoid cells to tumor-associated antigens (TAA) derived from two syngeneic C57BL/6J tumors was measured by [3H]thymidine incorporation. Peripheral blood cells were responsive to TAA from B16 melanoma and from BW10232 mammary carcinoma at both a high (10(-1) to 10(-3) mg/ml) and a low (10(-5) to 10(-6) mg/ml) concentration of antigen. While peripheral blood cells always responded to TAA, spleen cells and lymph node cells did not. When spleen and lymph node cells did respond, they sometimes responded at different concentrations of TAA than did the peripheral blood cells. Spleen cells generally responded to "low" concentrations of TAA, while lymph node cells responded to "high" concentrati-ns of TAA. These data suggest two subpopulations of lymphoid cells capable of response to TA.. Spleen cells from mice bearing the BW10232 mammary carcinoma became responsive to BW10232 TAA at low concentrations of antigen. Lymph node cells became responsive at high concentrations of BW10232 antigen. The response of both subpopulations to BW10232 TAA was amplified in peripheral blood cells. Spleen cells were 30 times more responsive in the tumor bearer than in normal animals, while lymph node cells were only 3 times more responsive. It is shown that lymphoid cells taken from different areas or "lymphoid compartments" do not always show similar responses and should not be considered equivalent in evaluating immune responses to tumor cells.

Tumor growth rate varies with age in lethargic mutant BALB/cGnDu mice.

BALB/cGnDu lethargic mutant mice suffer from an age-related temporary defect in their cell-mediated immune response which is "spontaneously" corrected in animals 7 weeks of age or older. Thus, mutants of different ages (3 to 4 weeks old and 7 to 9 weeks old) were used to compare tumor incidence, tumor growth rate, and host survival time of Harding-Passey (HP) melanoma, mKSA, and GI110(BK)B6D2Tu tumors. Normal littermates were used as controls. Only the HP tumor was successfully transplanted in all recipients. In the 3- to 4-week-old lethargic mutants, the HP tumor had an increased growth rate and decreased the mean lifespan of the mice, when compared to normal littermates, but only one mKSA and no GI110(BK)B6D2Tu tumors proved transplantable. In contrast, lethargic mutants 7 to 9 weeks old injected with the HP tumor survived longer than their normal littermates, and they did not accept either of the other tumors tested. These results corroborate the notion that lethargic mutant mice have a partially impaired anti-tumor cell-mediated immune response at 3 to 4 weeks of age, but that their anti-tumor response is enhanced at the time of their "spontaneous" correction of the immune deficiency. The need for future studies on the possible use of this model to study various human immunological and adrenal disorders is discussed.

Transfection and transformation of human and animal cells by recombinant DNA vectors: issues relevant to use of these substrates for the production of biologicals.

This paper reviews some of the issues relevant to the effect of oncogene or recombinant DNAs in both in vivo and in vitro models. Many studies of directly injected DNAs alone, with mediators of DNA uptake, or as the initiator in a multi-stage tumor progression model, showed that the DNAs were only rarely (if at all) tumorigenic. Conclusions from these and other in vitro experiments were that single oncogenes transfected into human cells did not generally convert those cells to a malignant phenotype, suggesting that additional genetic insult(s) or other factors were needed. These data, in concert with other observations and standard methods for product purification, imply that recombinant DNAs or oncogenes in cell lines pose little or no risk in the production of biologicals.

Potential risks of tumor virus subgenomes in the production of biologicals.

In this report, an attempt was made to describe what is presently known about the biological activities of tumor virus DNAs, their regulatory regions, and some factors which interplay with their oncogenic potential. In light of current knowledge, it is likely that the gene transfer risks of using continuous cell lines expressing known oncogenes, or cells containing oncogenic virus-recombinant DNA vectors, are minimal.

Subculture requirement for rescue of SV40 from 'nonrescuable' cell lines.

Virus could be rescued by cell fusion techniques from the H50, BRKSV (Bam I linear), 14B and 14B (1--4) cell lines, which contain the entire SV40 genome but have previously yielded negative results by this method. Virus could not be rescued from the T22 cell line, which was transformed by defective virus. The T-antigen-negative flat revertant cell line, 14B (1--4), converted to T-antigen-positive prior to rescue. Subculture and extensive monitoring for production of infectious virus appeared to be prerequisite for detecting virus rescue.

Rescue of infectious virus from permissive monkey cells containing simian virus 40 DNA fragments.

Permissive TC7 cells were separately transfected with simian virus 40 (SV40) EcoRI/Hap II A (74% genome) DNA fragments and EcoRI/Hap II B (26% genome) DNA fragments in the presence of DEAE-dextran. Fusion of the progeny of recipient cells receiving the A fragment, TC7 (SV40/74) cells, with TC7 (SV40/26) cells, which had received the B fragment, resulted in SV40 rescue. TC7 (SV40/74 + 26) cells, which had simultaneously received both complementary subgenomes, either spontaneously produced SV40 upon subculture or yielded virus upon treatment with iododeoxyuridine. In addition, fusion of rat cells containing the EcoRI/Hap II A fragment with TC7 (SV40/26) cells resulted in SV40 rescue. Cytopathology, V-antigen production, neutralization, and electron microscopy were parameters used to verify that the rescued virus was SV40. No infectious virus was produced when the combinations of cells fused did not total a complete SV40 genome equivalent.

A survey of intracellular Na+ and K+ of various normal, transformed, and tumor cells.

Intracellular concentrations of Na+ and K+ of various normal, transformed, and tumor cell cultures were analyzed by atomic absorption spectrophotometry. In all of the cultures analyzed there were markedly different concentrations in the transformed and tumor cells when compared to their normal counterparts. Although increased Na+ was often observed, there were no definitive correlations between absolute ion concentrations, or Na+:K+ ratios, and cell transformation.

Rescue of SV40 following transfection of TC7 cells with cellular DNAs containing complete and partial SV40 genomes.

Infectious SV40 virions could be rescued from permissive TC7 cells within one to three subcultures following cotransfection with two cellular DNAs, each containing a complementary portion of the SV40 genome. SV40 virions could also be rescued by transfection of TC7 cells with cellular DNAs from a variety of SV40 transformed cells containing complete genome equivalents but not from cells containing subgenomes alone or defective genomes. Infectious virus was not rescued if the transfecting DNA species was treated with DNAase or if the DEAE-dextran pretreatment of the recipient cells was omitted.

DNA-mediated transfection as a model for oncogenesis.

DNA transfer technology has greatly contributed to progress in understanding molecular biology and genetics. In recent years, great efforts have been expended to determine the oncogenic potential of single, defined genes or complex gene mixtures as a prelude to defining the role those genes may play in neoplastic transformation in vitro and tumor induction in vivo. This paper reviews the currently available DNA transfection techniques and their application toward understanding cancer initiation and progression, and how the in vitro and animal models may apply to human cancer.

The inability of in vitro transforming SV40 subgenomes to cause tumors in vivo.

Linear or subgenomic SV40 DNAs were transfected into cells from a variety of species (including rodent, dog, muntjak, and monkey) and injected subcutaneously into neonate Syrian hamsters for tumorigenicity testing. The 'early-region' subgenomes were capable of transforming cells in vitro. Complete genomes or complementary subgenomes could transform nonpermissive and semipermissive cells, were infectious for permissive cells, and induced tumors from which infectious virus could be rescued. Tumors were not formed in neonate hamsters upon injection with subgenomic SV40 DNAs, even those capable of transforming cells in vitro. These results suggested that SV40 tumor formation in vivo may require a complete genome.

Susceptibility of BALB/cGnDu mice to transplantable tumors, in vitro transformed cells, BK and SV40 viruses, and chemical carcinogens.

Various oncogenic agents were employed in tumor susceptibility studies of an inbred subline of BALB/cGn mice, BALB/cGnDu, which carries a mutant gene that results in spontaneous thymic involution in homozygous recessive individuals. Of 18 transplantable tumors, 3 in vitro transformed cell lines, 2 oncogenic papovaviruses and 3 chemical carcinogens evaluated, only the Harding-Passey amelanotic melanoma produced tumors in 100% of the normal adult mice injected. The S180 pleomorphic sarcoma produced tumors in 56% of normal adult mice. Although not tumorigenic in adults, the mKSA, Morris (rat) hepatoma No. 7777, and a human papovavirus - transformed human cell induced mouse tumor line [GI110(BK) B6D2 Tu] formed tumors in 100, 62 and 70%, respectively, of recipients inoculated as neonates. SV40 and human papovavirus BK virions, as well as sV40, BK, and spontaneous in vitro transformed BALB/cGnDu cells were not tumorigenic. Although the histocompatibility antigens of these animals have not been studied, no correlations could be made between the tumor susceptibility of these animals with regard to the commonly employed inbred line BALB/c nor with regard to the range of host susceptibility of the transplantable tumors. This inbred subline represents a new strain of mice whose genetic defect makes it useful for transplantation immunology and physiological genetics.