Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors.

While DNA damage response pathways are well characterized in cancer cells, much less is known about their status in normal cells. These pathways protect tumour cells from DNA damage and replication stress and consequently present potential therapeutic targets. Here we characterize the response of human telomerase reverse transcriptase (hTERT)-immortalized normal human urothelial (NHU) and bladder cancer cell lines to agents that disrupt the DNA damage response. Effects of replication and DNA damage response inhibitors on cell cycle progression, checkpoint induction and apoptosis were analysed in hTERT-NHU and bladder cancer cell lines. The primary signalling cascade responding to replication stress in malignant cells (ataxia telangiectasia-mutated (ATM) and Rad3-related-checkpoint kinase 1 (ATR-CHK1)) is not activated in hTERT-NHU cells after treatment with a replication inhibitor and these cells do not depend upon CHK1 for protection from apoptosis during replication stress. Instead, ATM signalling is rapidly activated under these conditions. Intriguingly, an ATM inhibitor suppressed S-phase checkpoint activation after exposure to replication inhibitors and stopped entry of cells into S-phase indicating G1 checkpoint activation. Consistent with this, hTERT-NHU cells treated with the ATM inhibitor showed increased levels of cyclin-dependent kinase inhibitor p19(INK4D), reduced levels of cyclin D1 and CDK4, and reduced phosphorylation of the retinoblastoma protein. In contrast, a bladder cancer cell line cotreated with ATM and replication inhibitors progressed more slowly through S phase and showed a marked increase in apoptosis. Taken together, our findings suggest that ATM and CHK1 signalling cascades have different roles in tumour and normal epithelial cells, confirming these as promising therapeutic targets.

DNA replication stress in CHK1-depleted tumour cells triggers premature (S-phase) mitosis through inappropriate activation of Aurora kinase B.

The disruption of DNA replication in cells triggers checkpoint responses that slow-down S-phase progression and protect replication fork integrity. These checkpoints are also determinants of cell fate and can help maintain cell viability or trigger cell death pathways. CHK1 has a pivotal role in such S-phase responses. It helps maintain fork integrity during replication stress and protects cells from several catastrophic fates including premature mitosis, premature chromosome condensation and apoptosis. Here we investigated the role of CHK1 in protecting cancer cells from premature mitosis and apoptosis. We show that premature mitosis (characterized by the induction of histone H3 phosphorylation, aberrant chromatin condensation, and persistent RPA foci in arrested S-phase cells) is induced in p53-deficient tumour cells depleted of CHK1 when DNA synthesis is disrupted. These events are accompanied by an activation of Aurora kinase B in S-phase cells that is essential for histone H3 Ser10 phosphorylation. Histone H3 phosphorylation precedes the induction of apoptosis in p53-/- tumour cell lines but does not appear to be required for this fate as an Aurora kinase inhibitor suppresses phosphorylation of both Aurora B and histone H3 but has little effect on cell death. In contrast, only a small fraction of p53+/+ tumour cells shows this premature mitotic response, although they undergo a more rapid and robust apoptotic response. Taken together, our results suggest a novel role for CHK1 in the control of Aurora B activation during DNA replication stress and support the idea that premature mitosis is a distinct cell fate triggered by the disruption of DNA replication when CHK1 function is suppressed.

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.

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.

Evidence for the early onset of aberrant promoter methylation in urothelial carcinoma.

There is evidence that carcinoma in situ (CIS) is the precursor of invasive urothelial carcinoma, a tumour characterized by frequent gene promoter methylation. The timing of altered DNA methylation is unknown in this pathway. Here we investigate gene methylation in 196 consecutive samples of normal urothelium, CIS, and tumours from 104 patients with both CIS and invasive urothelial carcinoma using quantitative methyl-sensitive polymerase chain reaction for six genes (p16, p14, E-cadherin, RARbeta2, RASSF1a, and GSTP1). Control normal urothelial samples from 15 patients with no history of urothelial carcinoma were also analysed. Immunohistochemistry established the expression of well-characterized CIS markers p53 and cytokeratin 20. Promoter methylation occurred frequently in both normal urothelium and CIS samples from patients with urothelial carcinoma, and increased with progression from normal to invasive urothelial carcinoma, at both specific loci (chi2 test: E-cadherin, p=0.0001; RASSF1a, p=0.003, RARbeta2, p=0.007, p16, p=0.024) and in general (methylation indices [t-test, p<0.0001]). Methylation was associated with cytokeratin 20 expression (t-test, p=0.004) and poor prognosis, and with increased progression to tumour death in patients whose CIS samples showed methylation, in comparison with those without methylation (log rank p<0.03). Promoter methylation occurs early in the urothelial carcinogenic pathway and appears to be a good biomarker of the invasive urothelial carcinoma phenotype.

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.

[Mutation of the FGFR3 oncogene is an independent and favorable prognostic factor for tumor-specific survival in patients with urothelial carcinoma of the upper urinary tract]. / FGFR3-Mutationen in Urothel-Carcinomen des oberen Harntrakts sind mit einer günstigeren Histopathologie und Klinik, aber nicht mit dem Mutator Phänotyp korreliert.

AIMS: Urothelial tumors of the upper urinary tract (UUTT) frequently display microsatellite Instability (MSI) and a distinct pathway of tumorigenesis resembling MMR-deficient colorectal cancers has been recognized for MSI-UUTT. For MSS-UUTT however oncogenic mechanisms as in bladder cancer (BC) are debated. Mutation of the oncogene FGFR3 has been linked to lower stage, lower grade and favourable clinical outcome in BC. The aim of this study was to evaluate FGFR3 mutation in MSI and MSS-UUTT. METHODS: 172 unselected UUTT were screened for MSI using the National Cancer Institute Consensus Panel and additional markers; FGFR3 status was studied using mutation analysis. Histopathological and clinical data were reviewed. RESULTS: Microsatellite status had no impact on histopathological or clinical outcome. 52/99 MSS, 10/22 MSI-low and 25/51 MSI-high UUTT displayed mut. FGFR3 respectively. Overall FGFR3 mutation was associated with favourable stage and grade (p <0.0001 and p <0.002), as 62.1% of mut. vs. 23.5 % of wt. FGFR3 UUTT were stage Ta and T1 and graded G3 in 25.3 % vs. 47.1% respectively. That effect depended on MS-status however, as FGFR3 mutation was related to lower stage (pTa and pT1) in MSS/MSI-L (mut 62.9 % vs. wt 18.7%; p <0.01) only as opposed to MSI-H (mut 60% vs. wt. 50%; p = 0,1) UUTTs and as FGFR3 mut UUTTs tended to display lower grade (G1 and G2) provided stable (mut 74,2 % vs. wt. 44.1%; p <0,01) as opposed to instable microsatellite status (mut 76 % vs. wt. 73 %; p = 0.7). There was no marked relation of MS-status or FGFR3 mutation to sex, age or tobacco-exposure; localization in the renal pelvis (p < 0.01) however was more prevalent in the FGFR3 mut group. As opposed to MSI-status FGFR3 mutation had a favourable impact on survival, as 24.1% vs. 54.2 % cancer related deaths occured in the mutated group (p <0.001); this effect was indendent on stage or MSI-status. Neither MSI- nor FGFR3-status had any influence on subsequent bladder tumors. CONCLUSIONS: FGFR3 mutation is frequent in UUTT and appears to be independent of MS-status; however it is related to significantly favourable histopathological parameters and clinical outcome in MSS cases. Thus our findings might back the notion, that MSS and MSI-UUTTs stem from different oncogenic pathways and that their differing molecular character might have some relevance. Further research is warranted to study the clinical behavior of these tumors and to evaluate a potential role for MS-status and FGFR3 as prognostic tools.

[Promoter methylation and microsatellite mutation reveals the clonal relationship of multiple urothelial carcinomas with mutator phenotype]. / Promotor-hypermethylierung und Mikrosatelliten-Instabilität in multifokalen Urothelkarzinomen mit Mutator-Phänotyp.

AIMS: The clonality of multiple urothelial carcinomas (UC) is subject to debate and affects treatment. Evidence derived from X-chromosome mosaicism and patterns of molecular alterations supports both a mono- and polyclonal relationship. In contrast to most UC, tumours with the mutator phenotype have frequent mutations in repetitive sequences (MSI) and promoter methylation. The aim of this study was to investigate the clonality of multifocal UC with MSI. METHODS: We have screened 400 UC for MSI and found it to occur in 1% of bladder and 15% of upper tract UC. Of these, 9 patients, whose tumours had MSI, developed or presented with multiple UC. A total of 32 UC (occurring over 0-6 years, 2-12 TCC per patient), 2 cases of CIS and 9 normal urothelial samples were screened for MSI at 17 loci and aberrant promoter methylation at 7 genes. RESULTS: In 8 of 9 patients, the pattern of microsatellite mutation and promoter methylation suggested that the multiple tumours had a clonal origin. Patterns of aberrant methylation in multiple tumours were more similar than microsatellite mutations, suggesting an earlier carcinogenic timing. MSI and promoter methylation were present in macroscopically normal urothelium from these patients. CONCLUSIONS: Aberrant promoter methylation occurs before microsatellite alteration in UC with mutator phenotype. The majority of recurrent UC with MSI are monoclonal in origin and macroscopically normal urothelium harbours multiple molecular abnormalities. Thus, at the time of apparently successful treatment, there is molecular evidence of residual tumour that subsequently develops into recurrent disease.

Proteomic analysis of voided urine after prostatic massage from patients with prostate cancer: a pilot study.

OBJECTIVES: Serum prostate-specific antigen measurements are widely used for the early detection of prostate cancer but lack specificity, thus warranting the search for additional biomarkers. METHODS: Two-dimensional gel electrophoresis followed by matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-TOF-MS) analysis was used to investigate the protein profiles of voided urine after prostatic massage from 6 patients with histologically confirmed prostate cancer and 6 age-matched patients with benign prostatic hyperplasia. RESULTS: The median number of protein spots per gel was lower in the urine from the patients with cancer (median 143 spots, range 118 to 163) than in the urine from those with benign prostatic hyperplasia (median 154 spots, range 142 to 209), although the difference was not statistically significant. MALDI-TOF-MS analysis identified six commonly expressed proteins: alpha-enolase, isocitrate dehydrogenase, beta-2-microglobulin, alpha-1-microglobulin, complex-forming glycoprotein HC, and PRO2044. Of the five protein spots seen in a subset of patients with cancer, one was identified as calgranulin B/MRP-14. Immunohistochemical staining of prostatic tissue showed greater expression of calgranulin B/MRP-14 in 2 of 7 well-differentiated, 1 of 12 moderately differentiated, and 0 of 8 poorly differentiated tumors relative to adjacent benign tissue; expression of calgranulin A/MRP-8, a heterodermic binding partner of calgranulin B/MRP-14, was absent. CONCLUSIONS: The role of urinary calgranulin B/MRP-14 as a potential novel marker for prostate cancer needs additional investigation.

Genetic instability and transitional cell carcinoma of the bladder.

The development of cancer occurs in a stepwise fashion, with each step representing the mutation in one of several key genes. However, the mutation rate of somatic cells is too low to account for the number of mutations required for a cell to undergo carcinogenesis. Thus, the development of genetic instability is a vital early step towards carcinogenesis. We review the evidence for genetic instability, with particular reference to transitional cell carcinoma of the bladder. Both microsatellite instability and chromosomal instability are present in this tumour, and we discuss their incidence and clinical implications.

Suppression of the radiation-sensitive phenotype of hamster irs1 and irs2 strains selected for resistance to 3-aminobenzamide.

PURPOSE: The radiosensitive hamster cell lines irs1 and irs2 have phenotypic similarities to cells defective in the early response to DNA-damaging agents as a result of mutations of the genes encoding poly(ADP-ribose)polymerase (PARP) or ataxia-telangiectasia mutated (ATM). Whether modification of PARP activity through selection of strains resistant to 3-aminobenzamide (3-AB) would affect the radiosensitive phenotype of irs1 and irs2 was examined. MATERIALS AND METHODS: 3-AB-resistant strains of irs1, irs2 and their parent line V79 were established and their sensitivity to DNA-damaging agents was measured. In some 3-AB-resistant strains, the radiation resistance of DNA synthesis and the induction of apoptosis were also assayed. Additionally, a number of aspects of PARP function were measured. RESULTS: Independently selected 3-AB-resistant strains of irs2 showed nearly complete suppression of radiation sensitivity, sensitivity to topoisomerase inhibitors, and radioresistant DNA synthesis. 3-AB-resistant strains of irs1 showed partial suppression of phenotype while 3-AB-resistant strains of V79 had no sensitivity changes. The induction of apoptosis in 3-AB-resistant strains of irs2 required substantially higher radiation doses than for irs2 itself. 3-AB-resistant strains had no detectable alteration of PARP level or cleavage following ionizing irradiation and there were no mutations in the PARP gene. CONCLUSIONS: Suppression of radiosensitivity associated with 3-AB resistance has important implications for mechanisms of tolerance to damage because it is able to override responses associated with specific genetic defects.

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.

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.

MSH3 deficiency is not sufficient for a mutator phenotype in Chinese hamster ovary cells.

In the yeast Saccharomyces cerevisiae, the mutS homolog protein products MSH3 and MSH6, each in cooperation with MSH2, play well-defined and specific roles in the repair of DNA mismatches and nucleotide loops. The discrete functions of the human homologs hMSH3 and hMSH6 are less clear and current evidence suggests that the substrate specificity of these proteins may be less strict. To determine the role of MSH3 in mammalian mismatch repair, we employed MSH3-deficient Chinese hamster ovary (CHO) cell lines. No significant changes in mutation rate were detected in the MSH3-deficient strain and there were no differences in sensitivity to DNA-damaging agents. Further analysis of hprt mutants did not show a MSH3-dependent shift in the mutant spectrum. Interestingly, thorough examination of four dinucleotide microsatellite regions revealed instability at only one locus in one of the MSH3-deficient cell lines. These data support the idea of a high degree of redundancy in the function of the MutS homologs MSH3 and MSH6, at least with respect to the control of microsatellite instability.

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.

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.

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.

The influence of a (GT)29 microsatellite sequence on homologous recombination in the hamster adenine phosphoribosyltransferase gene.

Several DNA sequence elements are thought to stimulate homologous recombination, illegitimate recombination, or both in mammalian cells. Some are implicated by their recurrence around rearrangement breakpoints, others by their effects on recombination of extrachromosomal plasmids. None of these sequences, however, has been tested on the chromosome in a defined context. In this paper we show how the adenine phosphoribosyltransferase locus in CHO cells can be used to study the recombinogenic potential of defined DNA sequences. As an example we have measured the effect on homologous recombination of a dinucleotide repeat, (GT)29, which has been shown to stimulate homologous recombination in extrachromosomal vectors 3-20 fold. On the chromosome at the adenine phosphoribosyltransferase locus, however, this sequence shows no capacity to stimulate recombination or to influence the distribution of recombination events.