• Differential survival of murine small and large intestinal crypts following ionizing radiation.

      Cai, W B; Roberts, Stephen A; Bowley, E; Hendry, Jolyon H; Potten, Christopher S; Potten, Christopher S; CRC Department of Epithelial Biology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1997-02)
      Radiation survival curves have been obtained for crypts in the small and large intestine of male BDF1 mice using the microcolony assay. Four regions of the small intestine and three regions of the large intestine were studied. One of the major objectives was to determine the optimal conditions for the microcolony assay in the large bowel. Various times post-irradiation were studied using different approaches and threshold criteria for surviving crypts. Small, but statistically significant, differences were observed along the length of the small intestine, but no differences were observed between different regions of the large intestine. There was a marked difference (p < 0.001) in the response in the large compared with the small intestine (D0 = 291 +/- 14 compared with 151 +/- 4 cGy respectively with corresponding extrapolation numbers of 18 +/- 4 and 377 +/- 65). The use of tritiated thymidine or vincristine helped in the identification of true survivors in the large bowel, but these approaches are not necessary in the small bowel. The longer the time after irradiation that the samples were fixed the easier it was to identify survivors in the colon. However, the longer the time the more animals died. The optimum compromise in these studies was to use partial-body irradiation (abdomen only) and to sample on the fifth day after irradiation. With these criteria the mid-colon survival curve had a D0 = 274 +/- 28 cGy and an extrapolation number of 22 +/- 10. The results can be considered in relation to published data on the levels of p53 and bcl-2 expression and apoptosis in murine small and large bowel.
    • Effect of bcl-2 deficiency on the radiation response of clonogenic cells in small and large intestine, bone marrow and testis.

      Hoyes, Katherine P; Cai, W B; Potten, Christopher S; Hendry, Jolyon H; CRC Experimental Radiation Oncology Group, Paterson Institute for Cancer Research, Manchester, UK. khoyes@picr.man.ac.uk (2000-11)
      PURPOSE: Overexpression of bcl-2 protects against radiation induced apoptosis in lymphohaematopoietic cell types in vivo, whilst bcl-2 deficiency radiosensitizes murine T-lymphocytes in vitro. However, there are few data regarding the influence of bcl-2 deficiency on the radiosensitivity of non-lymphoid cell types. The purpose of this study was to investigate the role of bcl-2 in the clonogenic radiation response of intestinal crypts, bone marrow progenitor cells and testicular stem cells. METHOD: Survival curves were obtained for each cell type from bcl-2 null (-/-), heterozygote (+/-) and wild type (+/+) mice. Crypt survival in the small and large intestine was assessed using the crypt microcolony assay. Committed haemopoietic progenitors were assayed using in vitro colony-forming cell (CFC) assays and survival of clonogenic spermatogonia was assessed by scoring regenerative tubules at 35 days post-irradiation. RESULTS: There was no difference in small intestine crypt survival between the three genotypes. In the colon, there was a tendency towards lower clonogen survival in the +/- and -/- animals. Haemopoietic in vitro CFC from -/- animals showed lower survival in comparison to +/+ mice, but spermatogonial stem cells were comparatively more radioresistant. CONCLUSIONS: Deficiencies in bcl-2 affect the radiation response of different cell populations in small but different ways. This may be due to variations between cells in their innate capacity for apoptosis, their dependence on different members of the bcl-2 family gene and their cell-cycle status and p53 expression.
    • The number of clonogenic cells in crypts in three regions of murine large intestine.

      Cai, W B; Roberts, Stephen A; Potten, Christopher S; CRC Department of Epithelial Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1997-05)
      Data are presented for the kinetics of repair of sub-lethal damage in the large intestine of mice. The results are based on experiments using the crypt microcolony assay with two equal sized doses which were delivered with a variable interval of time between the doses. These show that this split-dose repair was largely complete after 5 h, and that there were no significant differences between three regions of the large intestine. Overall the half-time for the repair was 2.3 +/- 0.8 h, and the maximum split-dose repair ratio (the proportion of damage recovered by splitting the dose into two fractions) was 22 +/- 2% and the mean recovery factor (the ratio of the number of surviving crypts using long interfraction intervals to that at zero time) was 11 +/- 2. The split-dose approach (Hendry 1979) using a 5 h interval has been used to estimate the number of clonogenic cells in large intestinal crypts. A range of single and paired doses between 7 Gy and 10.5 Gy were used. There were significant differences between the three regions of the large intestine, the caecum, mid-colon and rectum. The estimate of the number of clonogens also depended in a significant way on the dose of radiation used to make the estimate. At low doses large intestinal crypts contain between 5 and 10 clonogenic cells while if high doses were used they contain an estimated 16 to 36 clonogenic cells. Considerable similarity exists between the small intestine and the large intestine for; (a) the repair kinetics (b), the clonogenic estimates, and (c) their dependence on dose.
    • p53 deficiency sensitizes clonogenic cells to irradiation in the large but not the small intestine.

      Hendry, Jolyon H; Cai, W B; Roberts, Stephen A; Potten, Christopher S; CRC Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom. (1997-09)
      The role of p53 in the survival of irradiated crypts in the small intestine and three regions of the large intestine (cecum, mid-colon and rectum) was assessed by comparing the responses in p53 null, p53 heterozygous and wild-type mice. There was no difference in the levels of crypt survival in the small intestine between the three genotypes, although the rate of cell depletion and regeneration in the null mice appeared slower. In the large intestine, crypt survival was lowest in the null mice compared to the other genotypes, in particular after high doses. The levels of crypt survival in the heterozygotes were not significantly different from those in the wild-type mice. Hence the greater radioresistance of crypts in the colon than in the small intestine, reported previously by us and others using various other mouse strains, may be partly attributable to the presence of p53. This effect is not readily explained by current knowledge concerning the decreased p53 expression and the greater expression of the survival gene Bcl2 in the stem cell zone of crypts in the colon compared to those in the small intestine. Reduced repair associated with the lack of a G2-phase checkpoint delay, the predominant arrest point for intestinal cells, is a possible explanation for the decrease in survival.