Serial Transplantation of p53-Deficient Hemopoietic Progenitor Cells to Assess Their Infinite Growth Potential

Thirty-five years ago, Siminovitch et al. (Siminovitch L, Till JE, McCulloch EA. J Cell Com Physiol 64:23–32, 1964), using serially transplanted mouse spleens at 14-day intervals, observed a markedly progressive decline in the proliferative capacity of bone marrow (BM) cells, with the loss of cionogenicity by the fourth transplant generation. Using the same protocol, we assessed the proliferative capacity of p53-deficient mouse BM cells transplanted serially at the same 14-day intervals into lethally irradiated mice, which was a useful tool for understanding the characteristics of hemopoletic stem cells lacking solely the p53 gene function. BM cells from p53-deficient homozygous (p53–/–), p53-heterozygous (p53+/–), and wild-type (p53+/+) C57BL/6 mice were transplanted into lethally irradiated C57BL/6 recipients. Fourteen days later, the repopulated spleens were harvested, and 107 cells were retransplanted into secondary recipients. Serial transplantation was continued at 14-day intervals until hemopoietic repopulation failure. The number of heterozygous and homozygous p53-deficient spleen cells increased logarithmically up to the fourth and fifth passages, respectively, whereas wild-type spleen cells ceased to proliferate by the third passage. The number of macroscopic spleen colonies increased logarithmically until the third passage in recipients of heterozygous and homozygous p53-deficient cells, but ceased to grow by the second passage in recipients of wild-type cells. The numbers of heterozygous and homozygous p53-deficient colony forming units in spleen (CFUs-S) remained stable during the first four transplant generations, whereas that of wild-type CFUs-S decreased progressively from the first transplant generation onward. The clonogenicity of p53-deficient cells was lost when the number of CFUs-S per spleen decreased to below 10. This suggests that one out of 10 CFUs-S might be long-term repopulating cells (LTRCs), and that p53-deficient LTRCs may proliferate more rapidly than wild-type LTRCs. Longer passages that were possible in the p53-deficient groups were considered to be due to the faster cell cycle of the p53-deficient hemopoietic progenitor cells, as determined by bromodeoxyuridine incorporation with purging by UV light exposure, followed by hemopoietic colony assay (BUUV assay).

Reduction of Lung Metastases by Na[trans-RuCl4(DMSO)Im] is not Coupled With the Induction of Chemical Xenogenization

The effects of the treatment of tumor cells of MCa mammary carcinoma and TLX5 lymphoma with the ruthenium complex Na[trans-RuCl4(DMSO)lm] for several transplant generations were studied on tumor growth and metastases formation. On TLX5 lymphoma cells, treatment was performed in vitro prior to in vivo inoculation of tumor cells in intact or immunesuppressed mice. Either considering tumor take and growth or its capacity to invade the brain of the inoculated hosts, Na[trans-RuCl4(DMSO)lm] did not induce any significant modification. Conversely, in mice with MCa mammary carcinoma, the in vivo treatment of tumor cells in immunesuppressed hosts caused a progressive increase of DNA activity and, starting from the 4th transplant generation, a significantly increased susceptibility of lung metastasis formation to a further treatment in intact mice. These data seem to suggest that Na[trans-RuCl4(DMSO)Im] does not induce chemical xenogenization of tumor cells nor its repeated treatment induces resistance in tumor cells. Conversely, it appears that Na[trans-RuCl4(DMSO)lm] may select a tumor cell population which maintains its capacity to metastasise to the lung but with enhanced sensitivity to the antimetastatic properties of this compound.

Growth and Chemosensitivity of Human Head and Neck Cancers Implanted under the Kidney Capsule of Cyclosporine-Immunosuppressed Mice

Thirty-seven of 54 human squamous cell head and neck carcinomas were grown successfully as first transplant generation xenografts under the kidney capsule of conventional mice immunosuppressed by daily treatment with 60 mg/kg of cyclosporine. Eighteen different tumors were evaluated for chemosensitivity to cis-diamminedichloroplatinum (cisplatin), 5-fluorouracil, and methotrexate. Thirty-nine percent of the tumors responded to cisplatin, 19% to 5-fluorouracil, and 33% to methotrexate. This assay response is consistent with the clinical response of human squamous head and neck carcinoma to these drugs used as single agents. It is hoped that this model will become useful for new drug testing and, in certain cases, for selection of chemotherapy for patients with refractory tumors.

STUDIES ON THE HETEROLOGOUS IMMUNOGENICITY OF A METHANOL-INSOLUBLE FRACTION OF ATTENUATED TUBERCLE BACILLI (BCG)

A methanol-insoluble residue (MER) of phenol-killed attenuated tubercle bacilli (BCG), which has been reported previously to be capable of evoking heightened resistance to infection with antigenically unrelated microorganisms, was found to affect as well the resistance of highly inbred mice against tumor isografts. In most instances, the MER evoked heightened resistance against the tumor implants, but heightened susceptibility was the effect induced against two of the tumors tested, and no effect was elicited against one neoplasm. It is suggested that the heightened susceptibility occasionally produced by pretreatment with MER may also be of immunological nature, i.e. immunological enhancement. Treatment with MER was more effective when administered some time before tumor challenge than when given simultaneously with, or after, tumor implantation. The protective effects manifested against some tumors were of a high order, a significant number of animals rejecting the neoplastic implants, and were displayed even when several months elapsed between treatment and challenge. Living BCG and intact phenol-killed bacilli also evoked heightened resistance against some of the tumors tested, and in one experiment living BCG proved effective whereas MER did not. On the whole, however, MER was the most active (and least toxic, as shown previously) of the several tubercle bacillus preparations tested. MER elicited heightened reactivity against first transplant generation tumors as well as against tumors maintained for considerable periods of time by repeated animal passage, and against spontaneously arising as well as against induced neoplasms. The experimental parameters necessary to demonstrate maximal effects varied somewhat from tumor to tumor. In general, however, single intraperitoneal injections of small quantities of MER, of the order of 0.25 to 1.0 mg, afforded the best protection.