HDAC inhibitor confers radiosensitivity to prostate stem-like cells.

BACKGROUND: Radiotherapy can be an effective treatment for prostate cancer, but radiorecurrent tumours do develop. Considering prostate cancer heterogeneity, we hypothesised that primitive stem-like cells may constitute the radiation-resistant fraction. METHODS: Primary cultures were derived from patients undergoing resection for prostate cancer or benign prostatic hyperplasia. After short-term culture, three populations of cells were sorted, reflecting the prostate epithelial hierarchy, namely stem-like cells (SCs, α2ß1integrin(hi)/CD133(+)), transit-amplifying (TA, α2ß1integrin(hi)/CD133(-)) and committed basal (CB, α2ß1integrin(lo)) cells. Radiosensitivity was measured by colony-forming efficiency (CFE) and DNA damage by comet assay and DNA damage foci quantification. Immunofluorescence and flow cytometry were used to measure heterochromatin. The HDAC (histone deacetylase) inhibitor Trichostatin A was used as a radiosensitiser. RESULTS: Stem-like cells had increased CFE post irradiation compared with the more differentiated cells (TA and CB). The SC population sustained fewer lethal double-strand breaks than either TA or CB cells, which correlated with SCs being less proliferative and having increased levels of heterochromatin. Finally, treatment with an HDAC inhibitor sensitised the SCs to radiation. INTERPRETATION: Prostate SCs are more radioresistant than more differentiated cell populations. We suggest that the primitive cells survive radiation therapy and that pre-treatment with HDAC inhibitors may sensitise this resistant fraction.

Clustered base substitutions in CTP synthetase conferring drug resistance in Chinese hamster ovary cells.

Dominantly acting mutations that eliminate the allosteric regulation of CTP synthetase confer a form of multidrug resistance and a mutator phenotype to cultured Chinese hamster ovary cells. Mutations responsible for this phenotype have been identified in 23 independent strains selected for resistance to arabinosyl cytosine and 5-fluorouracil. All these mutations were due to base substitutions at seven sites within a highly conserved region of the ctps gene. This clustering should make it feasible to assess the role of such mutations in the development of drug resistance encountered in the treatment of malignant disease.

Apoptosis induced by replication inhibitors in Chk1-depleted cells is dependent upon the helicase cofactor Cdc45.

Checkpoint kinase 1 (Chk1) responds to disruption of DNA replication to maintain the integrity of stalled forks, promote homologous recombination-mediated repair of replication fork lesions, and control inappropriate firing of replication origins. This response is essential for viability as replication inhibitors trigger apoptosis in S-phase cells depleted of Chk1. Given the complex network of cellular responses controlled by Chk1, our aim was to determine which of these protect cells from apoptosis following replication stress. Work with cell-free systems has shown that RPA-ssDNA complex forms following replication inhibition through the uncoupling of replication and helicase complexes. Here we show that replication protein A (RPA) foci form in cells treated with replication inhibitors and that the number of foci dramatically increases together with hyperphosphorylation of RPA34 in Chk1-depleted cells in advance of the induction of apoptosis. RPA foci, RPA34 hyperphosphorylation, and apoptosis were suppressed by siRNA-mediated knockdown of Cdc45, an essential replication helicase cofactor required for both the initiation and elongation steps of DNA replication. In contrast, loss of p21, a negative effector of origin firing, stimulates both the accumulation of RPA foci and apoptosis. Taken together, these results suggest that the loss of control of replication origin firing following Chk1 depletion triggers the accumulation of the RPA-ssDNA complex and apoptosis when replication is blocked.

Conditional mutator phenotypes in hMSH2-deficient tumor cell lines.

Two human tumor cell lines that are deficient in the mismatch repair protein hMSH2 show little or no increase in mutation rate relative to that of a mismatch repair-proficient cell line when the cells are maintained in culture conditions allowing rapid growth. However, mutations accumulate at a high rate in these cells when they are maintained at high density. Thus the mutator phenotype of some mismatch repair-deficient cell lines is conditional and strongly depends on growth conditions. These observations have implications for tumor development because they suggest that mutations may accumulate in tumor cells when growth is limited.

Methylational urinalysis: a prospective study of bladder cancer patients and age stratified benign controls.

Tumour suppressor gene (TSG) methylation has been proposed as a diagnostic marker for urothelial cancer (UC). Here, we compare the frequency of urinary TSG methylation in young and elderly patients, with and without UC. Urine samples were obtained prospectively from 35 UC patients, 35 benign controls over the age of 70 years and 34 healthy volunteers under the age of 40 years. Methylation analysis was performed for eight gene promoters using quantitative methylation-specific PCR. Methylation was detected in urine DNA from all three patient groups. The highest frequencies were seen in UC patients. Significantly less methylation was present in control samples than UC cases for RASSF1a and APC (P < 0.034). The 'methylation index' and level of methylation was highest in the UC group and lowest in the young control group. A marker panel of RASSF1a, E-cad and APC generated a sensitivity of 69%, a specificity of 60% and a diagnostic accuracy of 86%. TSG methylation is detectable in urine DNA from patients with and without bladder cancer. The frequency and extent of methylation appears to increase with age and malignancy. The lack of tumour specificity suggests that further investigation is required before this test is introduced into clinical practice.

Sensitivity of a mutator gene in Chinese hamster ovary cell to deoxynucleoside triphosphate pool alterations.

The Thy- mutants of Chinese hamster ovary cells have a 5- to 10-fold elevated pool of deoxycytidine 5'-triphosphate (dCTP) and are auxotrophic for thymidine as an apparent consequence of a single mutation. thy is also a mutator gene, elevating the spontaneous rate of mutation 5- to 200-fold for at least two genetic markers. Previous experiments suggested that this mutator activity was caused by the elevated pool of dCTP in Thy- cells. To test this, the dCTP and deoxythymidine 5'-triphosphate (dTTP) pools were manipulated by altering the external concentration of thymidine in the growth medium. The rate of mutation at one genetic locus, ouabain resistance, was directly related to cellular dCTP content. At the highest level of dCTP the rate in one Thy- strain was approximately 200 times that of wild-type cells. However, the relationship between dCTP content and the rate of mutation at the ouabain locus was different for two mutator strains and wild-type cells. The rate of mutation at a second locus, thioguanine resistance, was increased approximately 10-fold over wild type regardless of the dCTP-dTTP pools. These experiments suggest that the mutator activity of thy is clearly related to dCTP content, but the dCTP level alone does not appear to be the cause of the mutator.

Increased mutational rates in Chinese hamster ovary cells serially selected for drug resistance.

Chinese hamster ovary cell populations exposed to the pressures of prolonged serial cultivation in cytotoxic drugs have increased mutational rates at independent genetic loci. Evidence suggests that the alterations generating these mutations may be independent of the lesions conferring drug resistance.

A novel pathway for transversion mutation induced by dCTP misincorporation in a mutator strain of CHO cells.

Intracellular imbalances of dCTP produce both T----C transitions and an unusual class of transversions (A----C) at the aprt locus of CHO cells. Our data suggest that this transversion pathway is the consequence of dCTP:T mispairs which are not efficiently proofread during DNA replication.

DNA sequence analysis of spontaneous mutations at the aprt locus of hamster cells.

To determine the nature of spontaneous mutational events in cellular genes in hamster cells, mutant adenine phosphoribosyltransferase (aprt) genes were cloned and the regions to which we mapped alterations were sequenced. A variety of nucleotide changes were found to occur in the 12 mutant genes analyzed. Most mutations were simple base-pair substitutions-transitions (both G X C----A X T and A X T----G X C) and transversions. The only multiple mutation was a simple transition next to a single-base-pair insertion. Of the 12 mutations, 4 were more complex, involving small deletions or duplications. Two of these were similar to previously described deletions in that they occurred between short direct sequence repeats. No hot spots were detected. Three independent mutations were characterized at one restriction endonuclease site, although no other mutations were detected in the nucleotides surrounding this site in other mutant strains. At a functional level, sequence changes were either in exons (resulting in missense and, in one instance, nonsense mutations) or at splicing sites.

Multiple dispersed spontaneous mutations: a novel pathway of mutation in a malignant human cell line.

We analyzed the nature of spontaneous mutations at the autosomal locus coding for adenine phosphoribosyltransferase in the human colorectal carcinoma cell line SW620 to establish whether distinctive mutational pathways exist that might underlie the more complex genome rearrangements arising in tumor cells. Point mutations occur at a low rate in aprt hemizygotes derived from SW620, largely as a result of base substitutions at G.C base pairs to yield transversions and transitions. However, a novel pathway is evident in the form of multiple dispersed mutations in which two errors, separated by as much as 1,800 bp, fall in the same mutant gene. Such mutations could be the result of error-prone DNA synthesis occurring during normal replication or during long-patch excision-repair of spontaneously arising DNA lesions. This process could also contribute to the chromosomal instability evident in these tumor cells.

Structural alterations of the aprt locus induced by deoxyribonucleoside triphosphate pool imbalances in Chinese hamster ovary cells.

Mutants induced at the adenine phosphoribosyl transferase (aprt) locus by dTTP or dCTP pool imbalances were examined for alterations in genomic DNA sequences. No observable changes were detected by Southern blot analysis of most mutant DNAs, suggesting induction of base pair alterations or other events below our level of detection (approximately 30 base pairs). However, in a few strains (11 from a total collection of 125 mutant cell strains), we were able to localize these events to restriction endonuclease recognition sequences when the mutations resulted in the loss or gain of a particular site. The distribution of lost or gained sites in aprt-deficient mutants induced by the two types of pool imbalances clearly varied, with those occurring in a mutator strain with increased dCTP clustering at one end of the aprt gene. Mutants induced by dTTP also revealed novel events: multiple restriction site modifications in a small region of the aprt gene in one mutant and a small (approximately 50 base pairs) insertion or duplication of DNA sequences. As in previous studies, very few deletion or insertion mutants were detected at the aprt locus. The significance of these findings in terms of the known biochemical and genetic consequences of these pool imbalances is discussed.

Loss of heterozygosity and base substitution at the APRT locus in mismatch-repair-proficient and -deficient colorectal carcinoma cell lines.

We determined the nature of mutations occurring at the autosomal APRT locus in mismatch-repair-proficient and -deficient colorectal carcinoma cell lines. The analysis of mutations that result in APRT deficiency in a mismatch-repair-deficient strain of DLD-1 heterozygous for this locus enabled us to measure the rate of loss of the wild-type gene through deletion, recombination, or gene conversion as well as the rate of point mutation. The overall rate of mutation at the APRT locus in DLD-1 was elevated 100-fold compared with the mismatch-repair-proficient colorectal carcinoma cell line SW620. Loss of heterozygosity (LOH) at APRT accounted for only 4 to 9% of mutant strains derived from DLD-1, indicating a rate for these types of events of 4 x 10(-7) to 9 x 10(-7). In SW620 the rate of LOH at APRT was about 10-fold higher. LOH was not found at polymorphic markers within the same chromosome subband as APRT, indicating that only a limited portion of the chromosome was affected by these alterations. Chromosome painting of SWS620 mutants revealed that the loss of APRT occurred together with a substantial portion of the long arm of chromosome 16. Differences in the nature of base substitutions at APRT (e.g., the proportion of mutations resulting from transitions or transversions) in these tumor cell lines were also detected. There was also an important similarity---the presence of a mutant APRT gene with multiple base substitutions that may be the result of some sort of error-prone DNA synthesis.

Alterations of gene structure in ethyl methane sulfonate-induced mutants of mammalian cells.

To determine the types of alterations in gene structure induced by DNA-alkylating agents, we analyzed the restriction enzyme cleavage patterns of adenine phosphoribosyltransferase gene sequences in mutant strains of Chinese hamster ovary cells deficient in this enzyme. Base pair changes as detected by loss of restriction enzyme sites were found, but no major internal gene rearrangements could be detected.

Increased rate of base substitution in a hamster mutator strain obtained during serial selection for gene amplification.

The pattern of mutations produced by a mutator gene (obtained during serial selection for amplification of the dihydrofolate reductase [dhfr] locus) shows a pronounced shift from that found in wild-type cells. The rate of certain types of base substitutions (particularly transitions) is dramatically increased, while gene rearrangements constitute a lower proportion of mutations. These data suggest a lower fidelity of the replication process in the mutator strain.

Expression of the endogenous O6-methylguanine-DNA-methyltransferase protects Chinese hamster ovary cells from spontaneous G:C to A:T transitions.

We have investigated whether the presence of a DNA repair enzyme, O6-methylguanine-DNA-methyltransferase (MGMT), affects the nature of spontaneous mutations in a mammalian cell line. We compared spontaneous mutations in the adenine phosphoribosyl transferase gene of a Chinese hamster ovary (CHO) cell line that expressed 14,000 MGMT molecules/cell with those in the parental CHO cells lacking this DNA repair activity. The mutation rate/cell/generation of the two CHO cell lines did not differ significantly. However, DNA sequence analysis of spontaneous mutations in the MGMT-proficient CHO cell line revealed a complex picture. No significant difference from the parental CHO cells was found in the number or type of deletions, frameshifts, multiple substitutions, or insertions. The frequency of G:C to T:A transversions was elevated in MGMT-proficient CHO cells. Expression of the enzyme considerably reduced G:C to A:T transitions (25% versus 8.3%). This latter result is the first evidence that this protein is active on an endogenous source of O6-methylguanine that is normally responsible for spontaneous G:C to A:T transition mutations.

Apoptosis induced by overexpression of hMSH2 or hMLH1.

Mutations of the mismatch repair genes hMSH2 and hMLH1 have been found in a high proportion of individuals with hereditary nonpolyposis colon cancer (HNPCC), establishing the link between mismatch repair and cancer. Tumor cell lines that are deficient in mismatch repair develop a mutator phenotype that appears to drive the accumulation of mutations required for tumor development. However, mutations of other mismatch repair genes such as hPMS2 can lead to a mutator phenotype, although inherited mutations of these genes are rare in HNPCC families. Here, we show that overexpression of hMSH2 or hMLH1 but not of hMSH3, hMSH6, or hPMS2 induces apoptosis in either repair-proficient or -deficient cells. Furthermore, primary mouse embryo fibroblasts derived from Msh2-deficient mice lose their ability to undergo apoptosis after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. These results suggest that the mismatch repair proteins hMSH2 and hMLH1 may be components of a pathway that influences apoptosis. We consider the possibility that loss of apoptosis as a result of hMSH2 or hMLH1 deficiency may be an additional factor in cancer predisposition in HNPCC.

Response of colon cancer cell lines to the introduction of APC, a colon-specific tumor suppressor gene.

The APC gene, mutations in which are responsible for the inherited colon cancer syndrome adenomatous polyposis coli (APC), is described as a tumor suppressor gene. A full-length, wild-type APC gene was introduced by transfection into three human colon carcinoma cell lines, each characterized for mutations at loci involved in colon tumor formation. The response of each cell line to the introduction of APC differed with the genotype of the cell line. Some of the cell clones derived from these transfections displayed altered morphologies; some showed suppression of tumorigenicity based on growth in soft agar and tumor formation in nude mice. One cell line, SW480, could not be stably transfected with the APC gene. These results provide the first direct evidence that the APC gene can alter the transformation properties of colon carcinoma cells.

Mutator phenotype in Msh2-deficient murine embryonic fibroblasts.

Embryonic fibroblast cell lines were established from mice deficient, heterozygous, or proficient for Msh2, one of the three known DNA mismatch repair genes involved in hereditary nonpolyposis colon cancer (HNPCC). Cell lines were established by transfection of primary mouse embryo fibroblasts with E7 and Ras oncogenes or mutant p53. Spontaneously immortalized cells derived from the primary cultures were also studied. To determine whether these cells developed a mutator phenotype similar to that found in colon cancer cells deficient in mismatch repair, we measured mutation rates, microsatellite instability, and sensitivities to a range of DNA-damaging agents. The mutator phenotype detected in the E7 and Ras or mutant p53-immortalized Msh2-/- mouse cells was similar to that found in human mismatch repair-deficient colorectal carcinoma cell lines. Mutation rates to ouabain resistance were increased 8-12-fold relative to lines from Msh2+/+ mice, and microsatellite instability was detectable in 12-18% of subclones derived from the Msh2-/- line but was undetectable in subclones developed from the Msh2+/+ line. Furthermore, E7 and Ras or spontaneously immortalized Msh2-/- cells were significantly more resistant to the cytotoxic effects of 6-thioguanine relative to Msh2+/+ cells. In contrast, these lines showed various responses to UV light and cis-platinum, suggesting that mismatch repair deficiency was not the sole determinant for sensitivity to these DNA-damaging agents. Particular attention was paid to the properties of cells heterozygous for the Msh2 mutant gene, which would mimic the situation of an HNPCC carrier. However, our studies failed to reveal any properties of these cells that might provide a growth advantage or predispose them for the acquisition of further mutations. This observation is consistent with the model that inactivation of the wild-type Msh2 allele is a critical step for tumorigenesis in HNPCC patients.

Microsatellite instability and the PTEN1 gene mutation in a subset of early onset gliomas carrying germline mutation or promoter methylation of the hMLH1 gene.

High-frequent microsatellite instability (MSI-H) was detected in two of the 80 gliomas examined, whlie the other 78 gliomas showed microsatellite stable (MSS) phenotype. Both of the two MSI-H tumors were glioblastomas which developed in teenage patients. One of the patient was diagnosed as having Turcot's syndrome and had a germline mutation in the hMLH1 gene. Loss of expression due to promoter methylation was selectively observed in the wild type allele of the hMLH1 gene in the tumor of this patient. The other patient had neither a family history nor a past personal history of malignancy. Although no mutation in the mismatch repair genes was detected in the tumor of this patient, the level of expression of the hMLH1 gene was markedly decreased and the promoter sequence of the gene was highly methylated. In the tumor of this patient, the PTEN1 gene, one of the genes carrying microsatellite sequences in their coding regions, was altered by a slippage mutation within five adenine repeat sequences. These findings indicate that the genetic or epigenentic inactivation of the hMLH1 gene is involved in a subset of early-onset gliomas and the PTEN1 gene could be a downstream target for mutation as observed in glioblastoma without MSI.

Insertion of unique and repetitive DNA fragments into the aprt locus of hamster cells.

Two insertion mutations occurring at the aprt locus of Chinese hamster ovary cells were analyzed at the nucleotide level by cloning and sequencing the mutant genes. The insertions are similar with respect to events at the target site in that both are accompanied by small deletions. The nature of the DNA introduced, on the other hand, is very different, a unique fragment in a spontaneously occurring mutant and a highly dispersed, repetitive fragment in a gamma radiation-induced strain. The inserts are small (285 and 58 base-pairs) and have none of the structural features or sequences related to putative mobile sequences in mammalian cells. The mechanism of transposition was further examined by cloning the unique donor fragment of the spontaneous mutant. These analyses revealed that the insert in the mutant gene was a precise duplicate of the donor DNA fragment.