Biology of marrow stromal cell lines derived from long-term bone marrow cultures of Trp53-deficient mice

Abstract

To investigate the effect of Trp53 (formerly known as p53) on stromal cells of the hematopoietic microenvironment, long-term bone marrow cultures were established from mice in which the Trp53 gene had been inactivated by homologous recombination (Trp53(-/-)) or their wild-type littermates (Trp53(+/+)). Long-term bone marrow cultures from Trp53(-/-) mice continued to produce nonadherent cells for 22 weeks, while Trp53(+/+) cultures ceased production after 15 weeks. There was a significant increase in the number of nonadherent cells produced in Trp53(-/-) long-term bone marrow cultures beginning at week 9 and continuing to week 22 (P < 0.02). The Trp53(-/-) cultures also showed significantly increased cobblestone island formation indicative of early hematopoietic stem cell-containing colonies beginning at week 10 (P < 0.01). Cobblestone islands persisted until weeks 15 and 22 in Trp53(+/+) and Trp53(-/-) cultures, respectively. Co-cultivation experiments in which Trp53(+/+) Sca1(+)lin- enriched hematopoietic stem cells were plated on Trp53(-/-) stromal cells showed increased cobblestone island formation compared to Trp53(-/-) Scal+lin- cells plated on Trp53(+/+) or Trp53(-/-) stromal cells. Radiation survival curves for clonal bone marrow stromal cells revealed a similar D0 for the Trp53(+/+) and Trp53(-/-) cell lines (1.62 +/- 0.16 and 1.49 +/- 0. 08 Gy, respectively; P = 0.408), and similar n (8.60 +/- 3.23 and 10.71 +/- 0.78, respectively) (P = 0.491). Cell cycle analysis demonstrated a G2/M-phase arrest that occurred 6 h after irradiation for both Trp53(+/+) and Trp53(-/-) stromal cell lines. After 10 Gy irradiation, there was no significant increase in the frequency of apoptosis detected in Trp53(+/+) compared to Trp53(-/-) marrow stromal cell lines. In the stromal cell lines, ICAM-1 was constitutively expressed on Trp53(+/+) but not Trp53(-/-) cells; however, a 24-h exposure to TNF-alpha induced detectable ICAM-1 on Trp53(-/-) cells and increased expression on Trp53(+/+) cells. To test the effect of Trp53 on the radiation biology of hematopoietic progenitor cells, the 32D cl 3 cell line was compared with a subclone in which expression of an E6 inserted transgene accelerates ubiquitin-dependent degradation of Trp53, thus preventing accumulation of Trp53 after genotoxic stress. The radiation survival curves were similar with no significant difference in the D0 or n, or in the percentage of cells undergoing apoptosis after 10 Gy irradiation between the two cell lines. Cells of the 32D-E6 cell line displayed a G2/M-phase arrest 6 h after 10 Gy, while cells of the parent line exhibited both a G2/M-phase arrest and a G1-phase arrest at 24 and 48 h. The results suggest a complex mechanism of action of Trp53 on the interactions between stromal and hematopoietic cells in long-term bone marrow cultures.