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

To investigate the effect of Trp53 (formerly known as p53) on stromal cells of the hematopoietic microenvironment, longterm bone marrow cultures were established from mice in which the Trp53 gene had been inactivated by homol ogous 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 productio n after 15 weeks. There was a significant increase in the number of nonadhe rent cells produced in Trp53(-/-) long-term bone marrow cultures beginning at week 9 and continuing to week 22 (P < 0.02). The Trp53(-/-) cultures als o 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. Go-cultivation experiments in which Trp53(+/+) Scal(+)lin(-) enriched hematopoietic stem cells were plated on T rp53-/- stromal cells showed increased cobblestone island formation compare d to Trp53(-/-) Scal+lin- cells plated on Trp53(+/+) or Trp53(-/-) stromal tells. Radiation survival curves for clonal bone marrow stromal cells revea led a similar D-0 for the Trp53(+/+) and Trp53(-/-) cell lines (1.62 +/- 0. 16 and 1.49 +/- 0.08 Gy, respectively; P = 0.408), and similar (n) over bar (8.60 +/- 3.23 and 10.71 +/- 0.78, respectively) (P = 0.491). Cell cycle a nalysis demonstrated a G(2)/M-phase arrest that occurred 6 h after irradiat ion for both Trp53(+/+) and Trp53(-/-) stromal cell lines. After 10 Gy irra diation, there was no significant increase in the frequency of apoptosis de tected in Trp53(+/+) compared to Trp53(-/-) marrow stromal cell lines. In t he stromal cell lines, ICAM-1 was constitutively expressed on Trp53(+/+) bu t not Trp53(-/-) cells; however, a 24-h exposure to TNF-a induced detectabl e ICAM-1 on Trp53(-/-) cells and increased expression on Trp53(+/+) cells. To test the effect of Trp53 on the radiation biology of hematopoietic proge nitor cells, the 32D cl 3 cell line was compared with a subclone in which e xpression of an E6 inserted transgene accelerates ubiquitin-dependent degra dation of Trp53, thus preventing accumulation of Trp53 after genotoxic stre ss. The radiation survival curves were similar with no significant differen ce in the D-0 or (n) over bar, or in the percentage of cells undergoing apo ptosis after 10 Gy irradiation between the two cell lines. Cells of the 32D -E6 cell line displayed a G(2)/M-phase arrest 6 h after 10 Gy, while cells of the parent line exhibited both a G(2)/M-phase arrest and a G(1)-phase ar rest at 24 and 48 h. The results suggest a complex mechanism of action of T rp53 on the interactions between stromal and hematopoietic cells in long-te rm bone marrow cultures. (C) 1999 by Radiation Research Society.