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

Abstract

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).

Cell cycle inhibitors in normal and tumor stem cells

“…It would be reasonable to expect the HSC phenotype of the p53À/À animal to be similar to that of p21À/À. In fact, in the absence of p53, HSC function has been reported to be significantly enhanced under stress conditions (Wlodarski et al, 1998;Hirabayashi et al, 2002) in a manner opposite to that found in the absence of p21 during long-term engraftment. Since one of the major cellular functions of p53 is to initiate cell death upon genotoxic stress, the enhanced function of HSCs in the absence of p53 suggests that increased survival may predominate over the accelerated proliferation of HSCs in some settings.…”

Using Mice to Examine p53 Functions in Cancer, Aging, and Longevity

“…p53 family members are likely to play a major role in preventing the emergence of cancer stem cells. Indeed, reductions in p53 dosage have been shown to enhance stem cell division as well as other functional attributes (Palacios et al 1996;Shounan et al 1996;Hirabayashi et al 2002;TeKippe et al 2003;Dumble et al 2007). Most recently, two studies have shown that p53 deficiency in hematopoietic multipotent progenitor cells and neural stem/progenitor cells contribute to enhanced proliferative capacity of these cells (Akala et al 2008;Zheng et al 2008).…”

The impact of altered p53 dosage on hematopoietic stem cell dynamics during aging

“…23 We hypothesized that reduced HSC functionality drove the diminished regenerative ability in the blood of p53 ϩ/m mice and that the opposite may be true for the p53-deficient animals, which may have increased stem-cell functionality. Studies of HSCs from young p53 null mice showed that these cells are more robust in their self-renewal and reconstitution functions, [26][27][28][29] but the retention or loss of these properties in older p53-deficient HSCs has not been examined.…”