Msh-2 suppresses in vivo mutation in a gene dose and lesion dependent manner.

Mice deficient for the mismatch repair (MMR) gene Msh2 show accelerated tumourigenesis and a reduced apoptotic response to DNA damage of methylation type. Here we examine the effect of mutation for Msh2 on in vivo mutation frequencies in the intestine as determined by loss of function at the Dolichos biflorus (Dlb-1) locus. Spontaneous mutation frequencies were scored in cohorts of ageing mice either wild type or mutant for Msh2. In mice less than 1 year old, mutation frequencies were only elevated in Msh2 null mice. However, beyond this age heterozygous Msh2 mice showed significantly higher mutation frequencies than controls. These findings implicate a gene dose dependent requirement for Msh2 in mutation suppression and prompted an analysis of young Msh2 mutants following exposure to DNA damage. Following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), Msh2 deficient mice show a reduced apoptotic response and an increase in mutation frequency. Heterozygotes did not differ from controls. Following exposure to cisplatin, no significant elevation was seen in mutation frequencies, even within homozygotes. This is particularly surprising given the association between cisplatin resistance and MMR deficiency. These findings therefore demonstrate a complex reliance upon functional Msh2 in mutation surveillance. We have identified three separate scenarios. First, where retention of both Msh2 alleles over an extended period of time appears critical to the suppression of spontaneous mutation; second, 3 weeks following exposure to MNNG, where only complete loss of Msh2 results in elevated mutation; and finally following cisplatin exposure, where induced levels of mutation are independent of Msh2 status.

Bodylifting: indications, technique and complications.

Bodylifting is often considered a high-risk surgical procedure. There is a widely held perception is that the operation is both time consuming and physically demanding for the surgeon, with, potentially, a long recovery and high complication rate for the patient. The senior author's experience of 16 consecutive bodylift procedures does not reflect this. Fourteen female patients and two male patients underwent Lockwood-type bodylifting procedures at two different hospitals over a 5 year period. Patients studied were unsuitable for a standard abdominoplasty either because of excess lateral abdominal tissue, or had undergone significant weight loss and developed redundant folds of skin in a circumferential pattern around the waistline. The average Body Mass Index (BMI) prior to surgery was 26.7. Mean surgical time and hospital stay was 4.2 hrs and 3.5 days respectively. No patient required blood transfusion. The follow up period was between 3 and 24 months. Levels of patient satisfaction were high and complications few. Only one patient required minor revisional surgery. No major complications were recorded. The commonest problem, seroma, developed in 4 out of 16 patients and was managed by simple aspiration. This study supports the effectiveness of the lower body lift as a procedure with the potential to produce an outcome unachievable by other means with a low incidence of minor complications.

DNA mismatch repair and colorectal cancer.

Colorectal cancer is the second commonest malignancy in the western world, accounting for 20,000 deaths in the U.K. per year. Over the last 10 years, great strides have been made in our understanding of the molecular controls governing the transition from normal mucosa, through adenoma, and finally to carcinoma. This review focuses on the recently discovered DNA mismatch repair pathway and its role in the development of both sporadic and an inherited form of colorectal cancer, namely hereditary non-polyposis colorectal cancer (HNPCC).

Susceptibility to apoptosis is differentially regulated by c-myc and mutated Ha-ras oncogenes and is associated with endonuclease availability.

Oncogenes and oncosuppressors can deregulate cell replication in tumours, and recently have been shown to influence the probability of apoptosis. The effects of human c-myc and mutated (T24) Ha-ras oncogenes on susceptibility to apoptosis were investigated by introducing them into immortalised rat fibroblasts. The resulting family of transfectants showed closely similar measures of proliferation, but widely divergent rates of apoptosis, differing by up to fifteen-fold, that correlated inversely with population expansion rates in vitro. T24-ras transfectants with moderate or high p21ras expression showed reduced apoptosis, and this was reversed by pharmacological inhibition of membrane localisation of p21ras by mevinolin. In contrast, c-myc stimulated apoptosis, and this was further enhanced by serum deprivation. Inducibility of effector proteins represents one possible mechanism of genetic control of the susceptibility to apoptosis, and its investigation showed that c-myc was associated with expression by viable cells of latent calcium/magnesium sensitive endonuclease activity characteristic of apoptosis. In contrast, endonuclease activity was not detected in viable cells of a T24-ras transfectant expressing high levels of p21ras. Thus, there appeared to be differential regulation of susceptibility to apoptosis, positively by c-myc and negatively by activated ras, and this was associated with availability of endonuclease activity. Genetic modulation of apoptosis in human neoplasms is likely to influence net growth rate, retention of cells acquiring new mutations and response to certain chemotherapeutic agents.

In vivo administration of O(6)-benzylguanine does not influence apoptosis or mutation frequency following DNA damage in the murine intestine, but does inhibit P450-dependent activation of dacarbazine.

Clinically relevant cancer chemotherapeutic alkylating agents such as temozolomide and dacarbazine induce apoptosis and are mutagenic via the formation of O(6)-alkylguanine adducts in DNA. The DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) functions by dealkylating such adducts and can thus prevent apoptosis and mutagenesis. In attempts to maximize the clinical effectiveness of these alkylating agents, inhibitors of AGT such as O(6)-benzylguanine (BeG) have been developed. We show here that within murine small intestinal crypt cells, BeG administration does not alter the apoptotic response to the direct-acting methylating agents N-methyl-N-nitrosurea (MNU), temozolomide and N-methyl-N'-nitro-N-nitrosoguanidine. Furthermore, we show that BeG pretreatment fails to elevate the mutation frequency at the murine Dlb-1 locus following exposure to MNU. Consistent with these results, we show that intestinal AGT activity is effectively abolished by administration of 100 mg/kg temozolomide, even in the absence of BeG. In contrast, pretreatment with BeG transiently abolished the apoptotic response to the methylating prodrug dacarbazine. Activation of dacarbazine to its reactive intermediate has previously been shown to be cytochrome P450 dependent and we show here that pretreatment of mice with the cytochrome P450 inhibitor metyrapone also inhibits dacarbazine-induced apoptosis. Thus BeG increases neither the prevalence of apoptosis nor mutation frequency in the murine small intestine, but is capable of inhibiting P450-dependent prodrug activation. The positive implication from this study is that BeG treatment may not exacerbate the toxic and mutagenic effects of methylating agents within normal cells, although it may engender other adverse reactions through the suppression of cytochrome P450-dependent processes.

Msh2 status modulates both apoptosis and mutation frequency in the murine small intestine.

Deficiency in genes involved in DNA mismatch repair increases susceptibility to cancer, particularly of the colorectal epithelium. Using Msh2 null mice, we demonstrate that this genetic defect renders normal intestinal epithelial cells susceptible to mutation in vivo at the Dlb-1 locus. Compared with wild-type mice, Msh2-deficient animals had higher basal levels of mutation and were more sensitive to the mutagenic effects of temozolomide. Experiments using Msh2-deficient cells in vitro suggest that an element of this effect is attributable to increased clonogenicity. Indeed, we show that Msh2 plays a role in the in vivo initiation of apoptosis after treatment with temozolomide, N-methyl-N'-nitro-N-nitrosoguanidine, and cisplatin. This was not influenced by the in vivo depletion of O6-alkylguanine-DNA-alkyltransferase after administration of O6-benzylguanine. By analyzing mice mutant for both Msh2 and p53, we found that the Msh2-dependent apoptotic response was primarily mediated through a p53-dependent pathway. Msh2 also was required to signal delayed p53-independent death. Taken together, these studies characterize an in vivo Msh2-dependent apoptotic response to methylating agents and raise the possibility that Msh2 deficiency may predispose to malignancy not only through failed repair of mismatch DNA lesions but also through the failure to engage apoptosis.