The Cosmetics Europe strategy for animal-free genotoxicity testing: project status up-date.

The Cosmetics Europe (formerly COLIPA) Genotoxicity Task Force has driven and funded three projects to help address the high rate of misleading positives in in vitro genotoxicity tests: The completed "False Positives" project optimized current mammalian cell assays and showed that the predictive capacity of the in vitro micronucleus assay was improved dramatically by selecting more relevant cells and more sensitive toxicity measures. The on-going "3D skin model" project has been developed and is now validating the use of human reconstructed skin (RS) models in combination with the micronucleus (MN) and Comet assays. These models better reflect the in use conditions of dermally applied products, such as cosmetics. Both assays have demonstrated good inter- and intra-laboratory reproducibility and are entering validation stages. The completed "Metabolism" project investigated enzyme capacities of human skin and RS models. The RS models were shown to have comparable metabolic capacity to native human skin, confirming their usefulness for testing of compounds with dermal exposure. The program has already helped to improve the initial test battery predictivity and the RS projects have provided sound support for their use as a follow-up test in the assessment of the genotoxic hazard of cosmetic ingredients in the absence of in vivo data.

Clinical relevance of genetic instability in prostatic cells obtained by prostatic massage in early prostate cancer.

We investigated whether genetic lesions such as loss of heterozygosity (LOH) are detected in prostatic cells obtained by prostatic massage during early diagnosis of prostate cancer (CaP) and discussed their clinical relevance. Blood and first urine voided after prostatic massage were collected in 99 patients with total prostate-specific antigen (PSA) between 4 and 10 ng ml(-1), prior to prostate biopsies. Presence of prostatic cells was confirmed by quantitative RT-PCR analysis of PSA mRNA. Genomic DNA was analysed for LOH on six chromosomal regions. One or more allelic deletions were found in prostatic fluid from 57 patients analysed, of whom 33 (58%) had CaP. Sensitivity and specificity of LOH detection and PSA free to total ratio <15% for positive biopsy were respectively 86.7 and 44% (P=0.002) for LOH, and 55 and 74% (P=0.006) for PSA ratio <15%. Analysis of LOH obtained from prostatic tumours revealed similar patterns compared to prostatic fluid cells in 86% of cases, confirming its accuracy. The presence of LOH of urinary prostatic cells obtained after prostatic massage is significantly associated with CaP on biopsy and may potentially help to identify a set of patients who are candidates for further prostate biopsies.

Prostate carcinoma risk and allelic variants of genes involved in androgen biosynthesis and metabolism pathways.

BACKGROUND: Ethnicity, when it is used to mean shared genetic inheritance within a group, has become one of the most important factors in determining prostate carcinoma risk. Genetic polymorphisms were hypothesized to be the probable explanation for differences in risk among ethnic groups. The authors evaluated the association between polymorphisms in genes involved in the androgen biosynthesis and metabolism pathway and the risk of prostate carcinoma. METHODS: Two hundred twenty-six patients with the pathologic diagnosis of sporadic prostate tumor and 156 healthy matched (age, ethnic group) male controls from a large epidemiologic cohort were genotyped for previously described polymorphisms in the androgen receptor (AR), 5alpha-reductase type II (SRD5A2), p450c17 (CYP17), and aromatase (CYP19) genes. The different polymorphisms in prostate carcinoma patients also were analyzed according to age of onset, preoperative prostate-specific antigen level, tumor stage, and tumor grade. RESULTS: The distribution of the tetranucleotide simple tandem repeat polymorphism (STRP) in intron 4 of CYP19 was significantly different in control and cancer patients (P = 0.012). The 171 allele and the 187 allele were associated with prostate carcinoma risk (P = 0.05 and P = 0.045, respectively). Conversely, no association was observed between prostate carcinoma risk and the other polymorphisms studied as follow: the CAG repeat in exon 1 of AR, the (TA)n dinucleotide repeat polymorphism in the 3' untranslated region, and the A49T or V89L substitutions in SDR5A2, the single base pair (bp) (a T to C transition) polymorphism that creates an additional Sp1-type (CCACC box) promoter site in CYP17. In prostate carcinoma patients, CAG repeats of AR, and TA repeats of SDR5A2 are associated with age of onset (P = 0.05 and P < 0.001, respectively). CONCLUSIONS: The association between the 171-bp allele of CYP19 and prostate carcinoma risk suggests that aromatase could be used as a new indicator for prostate carcinoma prevention in men of White French ethnogeographic origin. Conversely, it is possible that an individual carries both a high- and a low-risk marker (e.g., CYP17 A2 allele and V89L in SRD5A2) resulting in no overall difference in risk observed across the population. For these reasons, the development of a polygenic model, incorporating multiple loci from the individual genes may maximize the chance of identifying individuals with high-risk genotypes.

PCAP is the major known prostate cancer predisposing locus in families from south and west Europe.

To date four prostate cancer predisposing loci have been mapped: HPC1 (Hereditary Prostate Cancer 1) on 1q24-25, PCaP (Predisposing for Cancer Prostate) on 1q42.2-43, CAPB (Cancer Prostate and Brain) on 1p36, and HPCX on Xq27-28. We examined evidence for linkage to those loci in 64 families from south and west Europe. Genotyping of three (six for PCaP) markers encompassing the candidate regions were performed on 221 individuals including 159 affected patients. The resulting data were analysed using both parametric and non parametric linkage methods. No significant evidence of linkage to HPC1, CAPB, or HPCX was found either in the whole population or when pedigrees were stratified according to criteria specific to each locus. By contrast, results in favour of linkage to PCaP locus were observed with maximum multipoint NPL and HLOD scores of 2.8 (P = 0.0026) and 2.65 respectively. Homogeneity analysis performed with multipoint LOD scores gave an estimated proportion of families with linkage to this locus up to 50%. Particularly, families with an earlier age at diagnosis (< or = 65-years-old) contributed significantly to the evidence of linkage with a maximum multipoint NPL score of 2.03 (P = 0.024). Those results suggest that PCaP is the most frequent known locus predisposing to hereditary prostate cancer cases from families from south and west Europe.

Evaluation of androgen, estrogen (ER alpha and ER beta), and progesterone receptor expression in human prostate cancer by real-time quantitative reverse transcription-polymerase chain reaction assays.

Steroid hormones can have profound effects on prostate tumor development making it important to define steroid receptor expression in prostate tissues. For this purpose, androgen receptor (AR) and estrogen receptor (ER alpha and ER beta) expression was quantified in 12 clinically localized and 11 hormone-refractory sporadic prostate tumors, using real-time quantitative reverse transcription-PCR assays. To gain more insight into hormone-responsiveness, estrogen-regulated progesterone receptor (PGR) and androgen-regulated prostatic acid phosphatase (PAP) mRNA levels were also quantified. There is a decrease in expression of ER beta in both clinically localized and hormone-refractory tumors relative to normal prostate tissues. Moreover, hormone-refractory tumors display a decreased expression of ER alpha and an increased expression of AR. There is a positive association between ER alpha, ER beta, and PGR expression (P < 0.0001) and a negative association between AR and the androgen-regulated gene PAP expression in hormone-refractory tumors. Taken together, these data indicate that, although increased expression of the AR gene might play a key role in endocrine treatment failure, it cannot be considered as the sole actor of this unresolved dilemma, and abnormalities in ER alpha and/or ER beta expression may also modulate the growth response of prostate cancer to hormone withdrawal. Our results also suggest that ER alpha and ER beta expression status could be used to identify advanced prostate tumor patients who may respond to antiestrogen therapy.

Noninvasive detection of genetic instability in cells from prostatic secretion as a marker of prostate cancer.

We present a clinical and molecular study of a series of specific loss of heterozygosity (LOH) indicators which, together with PSA, increase the predictability of cancer in early prostate cancer patients. Considering a positive biopsy as the standard reference, the testing parameters for LOH testing are better than the PSA F/T ratio (<25%), suggesting that this noninvasive approach to detecting early prostate cancer could be very useful as a new tool to optimize the indications for iterative prostate biopsies.

VEGF overexpression in clinically localized prostate tumors and neuropilin-1 overexpression in metastatic forms.

Studies comparing tumor neovascularity with pathological findings suggest that angiogenesis contributes to the pathogenesis of prostate cancer. We have examined 42 primary sporadic prostate tumors at different clinical stages, together with 3 prostate cancer cell lines (DU145, PC3 and LNCaP), for expression of VEGF and the gene encoding the recently identified VEGF165 isoform-specific receptor neuropilin-1, by using a quantitative reverse transcription (RT)-PCR method. We also evaluated the VEGF transcription pattern. Upregulation of VEGF and neuropilin-1 was observed in 12 and 14 tumors, respectively. The VEGF165 isoform was slighly overrepresented in tumors that overexpressed VEGF. VEGF overexpression correlated with stage II disease (p < 0.05); neuropilin-1 overexpression correlated with advanced disease (p < 0. 01) and a high Gleason grade (p < 0.02). Our observations suggest that VEGF expression could be used as a prognostic marker in early-stage prostate tumors, whereas neuropilin-1 overexpression might be a marker of aggressiveness.

htert expression correlates with MYC over-expression in human prostate cancer.

Expression of the telomerase catalytic sub-unit (htert) constitutes a key step in the development of human cancer. Although htert regulation is still unclear, several studies suggest that c-myc may activate its expression. Prostate cancer is one of the most common malignancies among men in Western countries. Since de-regulated expression of myc as well as telomerase activation may contribute to the pathogenicity of this cancer, we investigated this pathway in prostate tumorigenesis. For this purpose, myc- and htert-mRNA expression was quantified in 33 sporadic prostate tumors using a real-time quantitative PCR assay based on TaqMan methodology. myc over-expression was observed in 19 (58%) of 33 tumors, whereas telomerase status evaluated by htert expression was observed in 22 (67%). There was no correlation between myc over-expression or htert expression level and tumor stage or Gleason grade. A significant association (p = 0.0024) was found between myc over-expression and elevated htert expression, indicating that the up-regulation of telomerase activity often observed in prostate tumors might be conferred through transactivation of htert by myc. It is likely that the ability of c-myc protein to stimulate expression of htert and thereby enhance telomerase activity represents an important step in prostate tumorigenesis.

Expression and mutational analysis of the MADR2/Smad2 gene in human prostate cancer.

BACKGROUND: Loss of heterozygosity (LOH) on chromosome arm 18q is common in sporadic prostate cancer and may be involved in cancer development through inactivation of tumor-suppressor genes (TSG). Recent identification, at 18q21.1, of MADR2/Smad2, a key component in transforming growth factor beta (TGFbeta)-family signaling pathways, led us to investigate the role of this gene in prostate tumorigenesis. METHODS: Sporadic primary prostate tumors from 25 patients with clinically localized tumors and 7 with metastatic forms were examined for MADR2/Smad2 mutations by using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis of cDNA, and for gene expression by quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: We detected no mutation in MADR2/Smad2 and no abnormal mRNA expression. CONCLUSIONS: Despite recent evidence indicating that MADR2/Smad2 acts as a tumor-suppressor gene, our findings suggest a limited role of this gene in prostate tumorigenesis, at least in the early stages. Another key tumor-suppressor gene may therefore be the main target of the observed LOH at 18q21.1.

Loss of heterozygosity at chromosome arm 13q and RB1 status in human prostate cancer.

Aberrations of the long arm of chromosome 13 are common in prostate cancer and were initially attributed to alterations of the RB1 gene in band q14 of the chromosome. However, prostate tumors generally yield normal p110RB1 nuclear staining despite loss of heterozygosity (LOH) at the RB1 locus. Our previous analysis of chromosome arm 13q showed allelic loss in 41% of primary prostate tumors. To refine our knowledge of 13q, we extended our previous LOH study by using more polymorphic markers to analyze more prostate tumors. Sixty human prostate carcinomas were screened for allelic loss on 13q by using 13 13q-specific markers. LOH on the long arm of chromosome 13 was found in 39 (65%) of the 60 tumors. Furthermore, 33 of these 39 tumors had evidence of allelic loss involving a region of 13q14 containing RB1. Because immunohistochemical assessment of pRb expression is controversial in prostate tumors, we used a quantitative reverse transcription polymerase chain reaction (RT-PCR) method to determine whether RB1 is the target tumor suppressor gene in this region. RB1 mRNA steady-state levels were determined in 12 prostate tumors preselected on the basis of presumed deletion at the RB1 locus and four prostate tumors without LOH at the RB1 locus; five normal prostate specimens were used as controls. One of the 12 assessable prostate tumors with presumed LOH at RB1 showed a corresponding decreased in RB1 mRNA expression, whereas none of the four tumors without LOH at RB1 locus showed such a decrease. This study, based on another technical approach, confirms that RB1 is not the main target of the observed LOH at 13q14.3, and raises the possibility that another tumor suppressor gene in this region plays a key role in prostate cancer.

Study of FHIT transcripts in normal and malignant breast tissue.

Cytogenetic and molecular studies have suggested that the 3p14.2 chromosome subband contains tumor suppressor genes involved in the pathogenesis of many types of human cancers. Recently, the FHIT (fragile histidine triad) gene was identified in this part of chromosome 3 as a candidate suppressor gene, and abnormal transcripts of this gene have been observed in various human tumors, including breast tumors. However, several investigators have challenged the involvement of FHIT in human cancers, especially because of discrepancies between data obtained with various PCR strategies and the observation that FHIT is alternatively spliced in normal tissues. We examined FHIT gene transcripts in a panel of normal (n = 27) and malignant (n = 33) breast tissue samples using single-stage PCR and two nested PCR strategies. In addition to a normal transcript, multiple variant transcripts were found at very low levels (<1% of the wild-type FHIT transcript) in the majority of the breast tumors, but also in adjacent normal breast tissues and normal breast tissue from women without cancer. These results do not support the involvement of the FHIT gene in breast tumorigenesis.

PTEN/MMAC1/TEP1 involvement in primary prostate cancers.

The PTEN/MMAC1/TEP1 gene, located at 10q23.3, is a tumor suppressor gene responsible for the familial cancer syndromes Cowden disease and Bannayan-Zonana syndrome, and is commonly somatically mutated in several types of cancers. Mutations of the PTEN gene have been found in prostate cancer cell lines and LOH at 10q22-24 in prostate tumors have also been described with a high frequency. To determine the role of this gene in prostate tumorigenesis, we therefore analysed 22 primary tumors for loss of heterozygosity (LOH) within the 10q22-23 region such that tumors hemizygous at those loci may be examined for somatic PTEN mutations. Losses of heterozygosity of at least one locus was found in 12 (55%) of the 22 tumors DNAs. Among these, six tumors exhibited allele loss in the interval between D10S1765 and D10S541 wherein lies the PTEN gene. We searched the entire coding region of PTEN for somatic mutations in these six tumors. One somatic mutation (17%), a 1 bp deletion, was detected in exon 7 of the gene, in one tumor, indicating that somatic mutations of the PTEN gene may occur in primary prostate tumors.

Genetic aspects of prostate cancer.

Despite its high incidence and mortality rate, the molecular mechanisms underlying the oncogenesis and progression of prostate cancer are still unclear. This review, based on recently published data, surveys the current state of knowledge of human prostate oncogenesis, dealing with genetic predisposition in familial clusters of prostate cancer, providing new information on the somatic genetic alterations, which have been approached in four ways (measurement of DNA content, cytogenetic analysis, in situ hybridization, and molecular analysis), and investigating the problems of androgen independence and intratumour heterogeneity in prostate tumours. Lastly, the potential clinical applications of the genetic alterations, which may become important in the near future, are addressed.

Predisposing gene for early-onset prostate cancer, localized on chromosome 1q42.2-43.

There is genetic predisposition associated with >=10% of all cancer of the prostate (CaP). By means of a genomewide search on a selection of 47 French and German families, parametric and nonparametric linkage (NPL) analysis allowed identification of a locus, on chromosome 1q42.2-43, carrying a putative predisposing gene for CaP (PCaP). The primary localization was confirmed with several markers, by use of three different genetic models. We obtained a maximum two-point LOD score of 2.7 with marker D1S2785. Multipoint parametric and NPL analysis yielded maximum HLOD and NPL scores of 2.2 and 3.1, respectively, with an associated P value of . 001. Homogeneity analysis with multipoint LOD scores gave an estimate of the proportion of families with linkage to this locus of 50%, with a likelihood ratio of 157/1 in favor of heterogeneity. Furthermore, the 9/47 families with early-onset CaP at age <60 years gave multipoint LOD and NPL scores of 3.31 and 3.32, respectively, with P = .001.

Molecular analysis of the FHIT gene in human prostate cancer.

A variety of studies suggest that the FHIT gene, which encompasses the fragile site at 3p14.2, is a candidate tumor suppressor gene in several forms of human cancer. To determine whether the FHIT gene is altered in prostate carcinomas, we examined 15 prostate tumors, four normal prostate tissue specimens and RNA from a pool of 62 normal human prostate tissues (Clontech) for the integrity of FHIT transcripts, using a robust single-stage PCR and a nested PCR method. In each case a major FHIT-specific full-length product was observed. Additional aberrantly sized products, which were more numerous in the nested PCR strategy, were present at a far lower level than the full-length transcripts in 14 of 15 tumors, three of four normal human prostate tissues and in the pooled normal prostate RNA. Sequence analysis revealed that these aberrant products corresponded to alternatively spliced FHIT transcripts, which were neither more numerous nor more prominent in the tumors than in the normal prostate specimens. Deletion at the FHIT locus was also evaluated by using three intragenic polymorphic markers (D13S1481, D3S1300, and D3S1234). Allelic loss was observed in two tumors, but these genomic alterations did not correspond to the aberrant FHIT transcripts. DNA analysis, furthermore, suggested that the tumor heterogeneity was not a likely explanation for presence of normal and alternatively spliced FHIT transcripts in the prostate tumors. In conclusion, we detected neither frequent loss of heterozygosity nor tumor specific transcripts of the FHIT gene in human prostate cancer.