Transfection and expression of mutant p53 protein does not alter the in vivo or in vitro growth characteristics of the AA/C1 human adenoma derived cell line, including sensitivity to transforming growth factor-beta 1.

Mutation of the p53 gene is thought to be a late event in human colorectal carcinogenesis, involved in the malignant conversion of the adenoma to the carcinoma. One of the questions that we hoped to address was whether, in vivo, a single mutational event in one p53 gene is sufficient to confer a significant growth advantage on a colonic epithelial cell. Such a growth advantage could result either from an increase in growth rate and/or loss of response to inhibitory growth signals naturally present in the colonic crypt. We therefore introduced the pC53-SCX3 143 (Val-Ala) p53 mutation into a non tumorigenic adenoma derived cell line, AA/C1, which contained a truncating APC mutation, activating K-ras mutation but was wild-type for the p53 protein. High levels of mutant p53 protein were detected in the pC53-SCX3 transfected AA/C1 cell lines but was found not to affect either the in vitro (colony forming efficiency, anchorage independence) or in vivo (tumorigenicity in nude mice) growth, when compared to vector control or the parental AA/C1 cell line. In addition, to test whether the cells become less sensitive to inhibitory growth factors, the response of the cell lines to the naturally occurring growth inhibitor TGF beta was also investigated. Even though TGF beta had previously been implicated in the control of growth of intestinal epithelium, expression of the mutant p53 protein did not affect the sensitivity of the parental AA/C1 cell line to TGF beta. Under the experimental conditions tested expression of the 143 (Val-Ala) p53 protein was unable to affect the in vitro or in vivo growth characteristics of the adenoma derived AA/C1 cell line. When compared to other studies, these results suggest that the genetic background of the individual recipient cell may greatly influence the effect of expression of a particular p53 mutation.

Molecular events including p53 and k-ras alterations in the in vitro progression of a human colorectal adenoma cell line to an adenocarcinoma.

The aim of the current study was to identify genetic abnormalities in human colorectal adenoma and carcinoma derived cell lines, and to determine whether the genetic changes which occur in vitro are relevant to the in vivo situation. Loss of 1p(33-35) region was shown to be the most common chromosome 1 abnormality and loss of heterozygosity (LOH) of the DCC gene and/or adjacent sequences was detected in all adenoma derived cells as well as the carcinoma cell lines. The level of p53 protein was also investigated as increased cellular p53 protein had previously been associated with mutation of the p53 gene. A further aim was to investigate genetic changes in our in vitro model of tumour progression, where the adenoma derived PC/AA cell line has previously been converted in vitro to two distinct tumorigenic phenotypes, producing either an adenocarcinoma or a mucinous carcinoma in athymic nude mice. Progression to the adenocarcinoma phenotype was shown to involve a specific chromosome 1 rearrangement, loss of both normal copies of chromosome 18 (although DCC gene sequences were retained), loss of the remaining wild type allele of k-ras resulting in homozygosity for the k-ras codon 12 mutation and increased cellular p53 protein as detected by SDS-PAGE Western blotting. The increase in p53 protein was shown not to be due to the acquisition of a mutation in the p53 gene. Interestingly, progression of the adenoma derived PC/AA cell line to the mucinous malignant phenotype did not involve any of these molecular rearrangements, suggesting that different genetically distinct pathways are involved in colorectal carcinogenesis. These studies show that the genetic changes in our in vitro model of human colorectal tumour progression are similar to those observed in in vivo studies.

The adenomatous polyposis coli protein and retinoblastoma protein are cleaved early in apoptosis and are potential substrates for caspases.

Apoptosis in human monocytic THP.1 tumour cells, induced by diverse stimuli, was accompanied by proteolytic cleavage of the adenomatous polyposis coli gene product (APC) and by sequential cleavage of the retinoblastoma susceptibility gene product (Rb). Cleavage of poly(ADP-ribose) polymerase (PARP), APC and the initial cleavage of Rb at the carboxy terminal region all occurred at a similar time, early in the apoptotic process. Subsequently, Rb underwent a secondary cleavage to 43 kDa and 30 kDa protein fragments. Two caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK) and acetyl-Tyr-Val-Ala-Asp chloromethyl ketone (YVAD.CMK), had markedly different effects on the induction of apoptosis. Z-VAD.FMK inhibited the primary and secondary cleavage of Rb, cleavage of APC and PARP, and apoptosis assessed by flow cytometry. In marked contrast, YVAD.CMK inhibited cleavage of APC and the secondary cleavage of Rb to the 43 kDa and 30 kDa protein fragments but did not inhibit the primary carboxy terminal cleavage of Rb, PARP proteolysis or apoptosis assessed by flow cytometry. These results suggest that different caspases are responsible for the cleavage of different substrates at different stages during the apoptotic process and that a caspase may either cleave APC directly or may be involved in the pathway leading to APC proteolysis. This is the first report suggesting that a cytoplasmic tumour suppressor gene (APC) may be cleaved by a caspase during apoptosis.

An optimised biphasic culture system for the generation of functional dendritic cells from patients with acute lymphoblastic leukaemia at presentation and in clinical remission.

We have tested the hypothesis that functional dendritic cells (DC) may be generated from patients with acute lymphoblastic leukaemia (ALL). We evaluated the production of DC from blast cells taken at presentation from nine children with ALL. Blast cells were expanded in serum-free medium supplemented with Flt3L, G-CSF, GM-CSF, IL-3, IL-6 and SCF for 7 days and subsequently stimulated with Flt3L, GM-CSF and TGF-beta for a further 14 days, with the addition of TNF-alpha for the final 48 h of culture. Cultured cells had the morphological appearance of DC and expressed the DC-associated antigens CD1A (range 2-87%) and CD83 (15-44%). Expression of the co-stimulatory molecules CD80 and CD86 was increased and the majority of these cells retained their expression of CD34 (73+/-4%) and HLA-DR (79+/-5%). Seven of the nine ALL had a leukaemia-specific abnormality and DC generated from five of these seven cases were derived from the leukaemic clone. Leukaemic DC derived from four HLA-A*02-positive ALL pulsed with CMV-associated peptides could induce significant proliferation of peptide-specific CD8+ T cells. This specificity was verified using tetrameric complexes of HLA class l/antigenic peptide. DC could also be generated from cells taken at times of complete remission of ALL and from normal controls using these culture conditions. These findings show that functional DC can be generated both from ALL blasts and from patients in remission; these might be utilised in future for immunotherapeutic strategies in the treatment of ALL.

A randomized comparison of psychological (cognitive behavior therapy), medical (fluoxetine) and combined treatment for women with premenstrual dysphoric disorder.

This study examines the relative effectiveness of cognitive behavior therapy (CBT) (ten sessions), fluoxetine (20 mg daily) and combined therapy (CBT plus fluoxetine) in women with premenstrual dysphoric disorder (PMDD). This was a randomized pragmatic treatment trial with three treatment cells. Treatment lasted for 6 months; a naturalistic follow-up was undertaken 1 year post-treatment. One hundred and eight women, satisfying the DSM-IV criteria for PMDD with 2 months' prospective confirmation were recruited into the study; sixty of these had completed 6 months of treatment and all measures before and after treatment. The main outcome measures were premenstrual scores on the Calendar of Premenstrual Experiences (COPE) and percentage of PMDD cases (DSM-IV diagnostic criteria). Significant improvement occurred in all three treatment-groups after 6 months' treatment, assessed by the COPE. Fluoxetine was associated with a more rapid improvement. There were no group differences in the percentage of DSM cases of PMDD post treatment, but at follow-up CBT was associated with better maintenance of treatment effects compared with fluoxetine. In conclusion, CBT and fluoxetine are equally effective treatments for PMDD, but the treatments have some differential effects that can be considered in treatment decisions. There appears to be no additional benefit of combining the treatments.

Loss of APC protein expressed by human colonic epithelial cells and the appearance of a specific low-molecular-weight form is associated with apoptosis in vitro.

APC (adenomatous polyposis coli) protein is differentially expressed in the normal colonic crypt and believed to be involved in colonic cell maturation. In this work we investigated whether expression of the APC protein is associated with cell death in colonic epithelial cells. We have previously reported an in vitro system to study apoptosis. Briefly, cells attached to the flask have a low frequency of apoptosis (1-3%), whereas cells that detach from the flask and float in the medium have a high proportion of apoptotic cells (36-96% depending on the cell line). The full-length 300-kDa or truncated APC protein, normally expressed by the attached cells (detected using the FE9 antibody), was found to be lost in the floating apoptotic cells in 8/11 colon tumour cell lines examined. In addition, the APC antibody FE9 detected a 90-kDa protein in the floating apoptotic cells of all cell lines investigated, which was not present in attached cells. Furthermore, loss of full-length APC and gain of the 90-kDa protein was observed in the apoptotic cells of 2 cell lines derived from other tissues: the SV40-transformed fibroblast cell line CMSV40fib and the lymphoblastoid B-cell line BJA-B. In cells repeatedly frozen and thawed, believed to induce necrotic cell death, full-length or truncated APC was also lost, though a 95-kDa protein distinct from that in apoptotic cells was observed. Specific loss of full-length or truncated APC (resulting in a 90-kDa protein in apoptotic cells but a 95-kDa protein in necrotic cells) is therefore associated with cell death. Our findings suggest a possible role for APC in cell survival.

The effect of different TP53 mutations on the chromosomal stability of a human colonic adenoma derived cell line with endogenous wild type TP53 activity, before and after DNA damage.

We examined the effect of loss of wild type TP53 activity on the chromosomal stability of a human colonic adenoma derived cell line (designated AA/Cl) by studying transfected variants which express different TP53 mutations. Using gross chromosomal aberrations as a measure of instability, we studied metaphase spreads of a vector control cell line (AA/PCMV) and variants expressing the 143(Val-Ala) mutation, which retain endogenous wild type TP53 activity, or the 273(Arg-His) TP53 mutation, which acts as a dominant negative. It was found that the proportion of cells with more than 4% aberrations was significantly greater in the AA/273p53/B cell line (an approximate 5-Fold increase) than in the vector control or the AA/143p53/A cell line. To investigate whether loss of TP53 dependent checkpoints also predisposed the cells to accumulate persistent chromosomal aberrations after DNA damage, cells were exposed to 5 Gy gamma radiation. Regardless of TP53 status, cells with radiation induced chromosomal damage were eliminated through a TP53 independent mechanism, suggesting that loss of TP53 activity did not permit the survival of these cells. In contrast, when exposed to low level gamma radiation (0.2 Gy), decreased wild type TP53 function and/or expression of mutant TP53 protein led to increased radioresistance (both in the non-dominant as well as the dominant mutant expressing cell lines). These findings suggest that loss of TP53 activity and/or acquisition of specific TP53 mutations can increase chromosomal instability and resistance to low level DNA damage in human colonic adenoma cells. This study emphasises the different biological consequences of individual TP53 mutations on the genotype of premalignant colorectal epithelial cells and subsequent implications for tumorigenic progression.

Biological consequences of the genetic changes which occur during human colorectal carcinogenesis.

Colorectal carcinogenesis is a complex multistage process and occurs through the accumulation of gene mutations in both oncogenes and tumour suppressor genes. Frequent genetic abnormalities include mutation of the familial adenomatous polyposis (APC) and/or the mutated in colorectal cancer (MCC) genes on chromosome 5q21, activation of K-ras and loss of the tumour suppressor genes p53 and DCC (deleted in colorectal cancer). In our laboratory we have developed human in vitro colonic cell culture model systems, to determine the biological consequences of these well characterised genetic changes, and how such changes can uncouple proliferation from differentiation and ultimately lead to the malignant phenotype.