Synthetic biology as driver for the biologization of materials sciences.

Materials in nature have fascinating properties that serve as a continuous source of inspiration for materials scientists. Accordingly, bio-mimetic and bio-inspired approaches have yielded remarkable structural and functional materials for a plethora of applications. Despite these advances, many properties of natural materials remain challenging or yet impossible to incorporate into synthetic materials. Natural materials are produced by living cells, which sense and process environmental cues and conditions by means of signaling and genetic programs, thereby controlling the biosynthesis, remodeling, functionalization, or degradation of the natural material. In this context, synthetic biology offers unique opportunities in materials sciences by providing direct access to the rational engineering of how a cell senses and processes environmental information and translates them into the properties and functions of materials. Here, we identify and review two main directions by which synthetic biology can be harnessed to provide new impulses for the biologization of the materials sciences: first, the engineering of cells to produce precursors for the subsequent synthesis of materials. This includes materials that are otherwise produced from petrochemical resources, but also materials where the bio-produced substances contribute unique properties and functions not existing in traditional materials. Second, engineered living materials that are formed or assembled by cells or in which cells contribute specific functions while remaining an integral part of the living composite material. We finally provide a perspective of future scientific directions of this promising area of research and discuss science policy that would be required to support research and development in this field.

Duration of neoadjuvant androgen deprivation therapy before radical prostatectomy and disease-free survival in men with prostate cancer.

There is little evidence that neoadjuvant androgen deprivation therapy (ADT) of 3 months' duration before radical prostatectomy (RP) favorably influences disease-free survival. However, recent data suggest that prolonged treatment may improve outcome. We conducted a prospective cohort study to determine whether ADT of either standard or prolonged duration before RP influences the risk of prostate-specific antigen (PSA) failure. We followed 756 men treated for prostate cancer by RP between 1991 and 1998 in Quebec City. Of these, 240 received combined neoadjuvant ADT for either /=93 days (111 men), and 516 were treated by RP alone. Multivariate Cox regression was used to estimate the hazard ratios (HR) of PSA failure (>0.3 ng/mL) associated with treatment regimen controlling for age, clinical stage, grade, and initial PSA level. The median duration of follow-up was 4 years. Compared with men treated by RP alone, those who received neoadjuvant ADT for >/=93 days had an HR of PSA failure of 0.60. The inverse association with the risk of PSA failure became statistically significant from the third year on, reached its greatest magnitude after 4 years, and was still present 8 years after RP. No association was observed for ADT of

Expression of p21 predicts PSA failure in locally advanced prostate cancer treated by prostatectomy.

The management of prostate cancer is based on several clinicopathological criteria. The ability to determine the tumor's biological potential is one goal of tumor markers in order to identify patients who may require more intensive treatment strategies. The purpose of our study was to determine if p21/(WAF1/CIP1) expression can predict biochemical failure in patients with locally advanced prostate cancer treated by radical retropubic prostatectomy (RRP). We used immunohistochemistry to analyze patterns of p21 expression in a population of 296 patients with locally advanced (pT3) prostate cancer treated by RRP. Results were correlated with clinicopathological parameters and time to PSA failure. For the entire cohort of 296 patients, after adjustment for prognostic factors, p21 expression was associated with an increased risk of PSA failure (relative risk [RR] = 1.48) of statistical significance at a median follow-up of 54.5 months. Other parameters that independently predicted the risk of PSA failure included lymph node metastasis and seminal vesicle involvement. Because neoadjuvant hormonal therapy (NHT) is known to lead to involutional changes in prostatic carcinoma, we performed multivariate analyses after stratifying for NHT prior to surgery. Among the 172 patients treated by RRP alone, p21 expression was an independent predictor of PSA failure (RR = 2.30), as were lymph node metastases (RR = 3.19) and pathological grade 5-7 and 8-10 (RR = 2.87 and 3.50, respectively). p21 over-expression is an independent predictor of PSA failure in pT3 patients treated by radical prostatectomy, especially if they did not receive NHT. This tumor marker may help clinicians identify patients who may require adjuvant treatment strategies following radical prostatectomy.

Can biological markers predict recurrence and progression of superficial bladder cancer?

Biological markers that are predictive of recurrence and progression of superficial bladder tumors must provide additional information to that provided by multiplicity, size and grade. Field anomalies in normal appearing urothelium of patients with papillary superficial transitional cell carcinoma have been associated with tumor antigens and chromosome 9 deletions. Also, primary tumors with chromosome 9 deletions are associated with a higher risk of recurrence. Abnormal expression of p53, p21 and Ki-67 cell cycle markers have little predictive value for recurrence. However, p53 overexpression or mutation and decreased expression of p27 are associated with cancer progression and survival. New markers, such as mutations in the fibroblast growth factor receptor 3 gene (found in 30% of tumors), anomalies of the PTEN gene and vascular endothelial growth factor expression, may have potential and require further evaluation. Molecular fingerprints of superficial tumors with distinct clinical behavior are being rapidly unravelled. Large-scale clinical studies are urgently needed to provide supportive evidence for their incorporation in clinical management.

Tumorigenic pathways in low-stage bladder cancer based on p53, MDM2 and p21 phenotypes.

Our aim was to determine whether the pattern of expression of the interrelated proteins p53, MDM2 and p21 could shed light on the etiopathogenic mechanisms of superficial bladder tumors. Protein expression was detected by immunohistochemistry (IHC) using monoclonal antibodies (MAbs) Pab 1801 for p53, IF2 for MDM2 and EA10 for p21 on 269 newly diagnosed bladder tumors (214 pTa and 55 pT1; 93 grade I, 145 grade 2 and 31 grade 3). While no p21 immunoreactivity was found in normal urothelium, 85% of tumors were strongly p21-positive. MDM2 was overexpressed in 44% of tumors, almost all being also positive for p21. p53 was overexpressed in 20% of cases: 66% of p53-positive tumors were also MDM2 positive, compared with only 38% of p53-negative tumors. p53 mutations were studied by direct DNA sequencing in a subset of 24 high-grade tumors. Both MDM2 and p21 were less frequently expressed in p53 mutated tumors compared with tumors with a wild-type gene. Distinct phenotypes were correlated with the frequency of poorly differentiated (grade 3) tumors. The most common phenotypes were p21+/MDM2-/p53- and p21+/MDM2+/p53- observed in 38% and 29% of tumors, respectively. Grade 3 tumors were found in 4% and 8% of these 2 groups, in contrast with 30% frequency in p21+/p53+ tumors (p = 0.002) regardless of their MDM2 phenotype. Four of the 5 (80%) tumors that were p53-positive but negative for p21 were grade 3. Our data suggest that several tumorigenic pathways for superficial bladder tumors can be reflected by the expression pattern of these 3 proteins.

Neoadjuvant hormonal therapy before radical prostatectomy and risk of prostate specific antigen failure.

PURPOSE: To date there is little information on the long-term effect of neoadjuvant hormonal therapy on prostate cancer progression. We performed a prospective study to determine whether patients with prostate cancer receiving neoadjuvant hormonal therapy before radical prostatectomy (hormonal therapy group) have a lower risk of prostate specific antigen (PSA) failure than those treated with radical prostatectomy alone (prostatectomy group). We also evaluated whether type of neoadjuvant hormonal therapy and duration were associated with the risk of PSA failure. MATERIALS AND METHODS: We followed 680 men initially treated for prostate cancer with radical prostatectomy between January 1988 and December 1997 at our university hospital. Of the patients 292 received neoadjuvant hormonal therapy. Median followup was 38 months. Cox regression analysis was used to assess the association between neoadjuvant hormonal therapy and PSA failure (greater than 0.3 ng./ml.) controlling for age, clinical stage, grade, initial PSA and adjuvant therapies. RESULTS: Surgical margins were positive less often in the hormonal therapy (25%) than the prostatectomy (47%) group (p = 0.0001). PSA failure was observed in 163 patients and the 5-year failure rate was 33%. No difference in risk of PSA failure was observed overall between the hormonal therapy and prostatectomy groups (hazards ratio 0.94, 95% confidence interval 0.68 to 1.30). Treatments with antiandrogen alone for any duration, and those combining antiandrogen and luteinizing hormone-releasing hormone analogue for 3 months or less were not associated with improved survival. However, patients receiving combined therapy for more than 3 months had a significantly lower risk of PSA failure than those treated with radical prostatectomy alone (hazards ratio 0.52, 95% confidence interval 0.29 to 0.93). CONCLUSIONS: Prolonged neoadjuvant hormonal therapy combining antiandrogen and luteinizing hormone-releasing hormone analogue may improve disease-free survival after radical prostatectomy.

Prognostic value of the proliferative index determined by Ki-67 immunostaining in superficial bladder tumors.

The biological behavior of urothelial carcinomas remains unpredictable. The objective of this study was to determine the prognostic value of Ki-67 index in superficial papillary bladder tumors and to correlate it with the S-phase fraction (SPF) measured by flow cytometry. Three hundred nineteen patients with newly diagnosed superficial (pTa, pT1) bladder tumors were included between September 1990 and April 1992. Patients with bladder carcinoma in situ alone were excluded. We observed 255 pTa tumors and 64 pT1 tumors, whereas 111 lesions were classified as grade G1 and 208 as grade G2-G3. Ki-67 immunostaining was performed on paraffin-embedded material using a 3-step immunoperoxidase procedure with the murine monoclonal antibody MiB1. The relation between Ki-67 expression and prognostic variables (stage, grade, tumor size, multifocality, age, and sex) was investigated by the chi-square test. Cox regression was used to describe the association between Ki-67 and tumor recurrence in 308 patients with follow-up while adjusting for potentially confounding prognostic variables. The frequency of high Ki-67 expression (> or =10%) increased with stage (P = .005) and grade (P = .001), but not with tumor size or multifocality. Two hundred one patients experienced tumor recurrence in a median follow-up of 68 months. Stage, grade, tumor size, and multifocality were all independent predictors of recurrence. Ki-67 index greater than 10% was found to be an independent predictor of tumor recurrence among patients with tumors larger than 3 cm in diameter (HR = 2.05, CI = 1.18-3.55), but not those with smaller size tumors. With regards to the DNA index, a significant but weak correlation was observed between Ki-67 expression and the SPF (Spearman's correlation coefficient = 0.23, P = .004). In addition, aneuploid tumors had significantly higher expression of Ki-67 (22.5%) than diploid tumors (10.1%) (P = .0006). Moreover, patients with DNA aneuploid bladder tumors were more likely to have more than 10% Ki-67-positive cells than those with diploid tumors. In patients with newly diagnosed pTa or pT1 bladder tumors, a Ki-67 index above 10% is an independent predictor of shorter time to recurrence only in those with tumors larger than 3 cm.

Deletions of the INK4A gene in superficial bladder tumors. Association with recurrence.

The INK4A and the INK4B genes map to chromosome 9p21, an area frequently deleted in bladder neoplasms. In addition to the p16 protein, the INK4A encodes for a second product, termed p19(ARF). We analyzed tissues from 121 patients with initial Ta and T1 tumors. Deletions of the INK4A gene were observed in 17 of 121 (14.1%) cases. Point mutations were identified in 2 of 64 (3.1%) tumors. The INK4A-exon 1beta and the INK4B gene were codeleted with INK4A in all of the homozygously deleted cases analyzed. The p16 promoter underwent de novo methylation in 7 of 47 (14.9%) evaluable cases. The p16-positive phenotype was observed in 18 of 56 (32%) evaluable cases. p16 negative phenotype correlated with deletion and methylation status. A statistically significant association between INK4A homozygous deletions and tumor size was observed (P = 0.003). Patients bearing tumors with INK4A homozygous deletions had a lower recurrence-free survival (P = 0.040) than those with wild type INK4A. In conclusion, deletions and methylation of the INK4A gene occur frequently in superficial bladder tumors. However, only those deletions that affect both the p16 and the p19(ARF), deregulating both the pRb and p53 pathways, correlated with clinicopathological parameters of worse prognosis.

Male urethral carcinoma: analysis of treatment outcome.

OBJECTIVES: To evaluate our experience with primary carcinomas of the male urethra and to analyze the impact of tumor variables and treatment on overall, disease-specific, local recurrence-free, and metastasis-free survival. METHODS: Between 1958 and 1996, we identified 46 men with primary carcinoma of the bulbar and anterior urethra. The median follow-up was 125 months (1 to 336). The patients were stratified by stage, nodal status, histologic type, treatment, type of surgery, site of disease, year at diagnosis, and smoking status. RESULTS: The overall survival and disease-specific survival rates at 5 years were 42% and 50%, respectively. The recurrence-free survival and metastasis-free survival rates at 5 years were 51% and 56%, respectively. The overall survival rate was 83% for superficial disease versus 36% for invasive tumors. The overall survival rate was 26% for tumors of the bulbar urethra versus 69% for tumors of the anterior urethra. CONCLUSIONS: Current modalities of treatment are ineffective for local control and survival. New treatment strategies are needed for urethral cancer.

Predictive value of cell cycle markers p53, MDM2, p21, and Ki-67 in superficial bladder tumor recurrence.

The aim of the study was to determine whether the expression of the cell cycle markers p53, MDM2, p21, and Ki-67 was predictive of superficial bladder cancer recurrence and to compare the relative predictive power for tumor recurrence of a cell cycle index based on the number of abnormally expressed cell cycle markers with a clinicopathological index based on primary clinical tumor characteristics. The expression of p53, MDM2, and p21 proteins and the value of the Ki-67 index were analyzed for 244 patients. One hundred ninety-four lesions were determined to be superficial papillary tumors (pTa), whereas 50 tumors invaded the lamina propria (pT1). Tumor grade was noted low (grade 1) in 83 cases and high (grades 2-3) in 161 cases. An avidin-biotin peroxidase method was performed using monoclonal antibodies against p53, MDM2, p21, and Ki-67 antigens after antigen retrieval treatment of formalin-fixed specimens. The cell cycle marker index was created using the number of abnormally expressed cell cycle markers according to the following cutoff points: p53 (>5%), MDM2 (>20%), p21 (<5%), and Ki-67 (>10%). The clinicopathological index was created using the following adverse tumor characteristics: grades G2-G3, stage pT1, multifocality, and diameter of tumors > 3 cm. Cox regression models were used to calculate the relative risks and their 95% confidence intervals associated with disease recurrence for the clinicopathological index and the cell cycle marker index. The chi2 test was performed to describe the correlation between the Ki-67 index and p53, MDM2, and p21 protein expression. Kaplan-Meier survival curves were generated to demonstrate the disease-free survival according to these two prognostic indexes. The clinicopathological index was a strong, independent predictor of disease recurrence where tumors with three or four adverse tumor characteristics at initial resection had over four times the risk of recurrence than tumors with no risk factors (P for trend = 0.0001). A strong correlation was observed between the Ki-67 index >10% and both MDM2 and p21 proteins. MDM2 was overexpressed in 106 tumors (43%), and p53 was overexpressed in 47 (19%); Ki-67 was >10% in 171 cases (70%). Thirty-nine tumors (16%) were p21 negative. The risk of recurrence increased slightly with the number of abnormally expressed cell cycle markers, but when the clinicopathological index was taken into account in multivariate analysis, the cell cycle marker index was not predictive of disease recurrence (P for trend = 0.72). The cell cycle markers studied provided no added prognostic information on disease recurrence after initial resection of papillary superficial tumors when the clinicopathological parameters were taken into account.

Genotypic and phenotypic characterization of the histoblood group ABO(H) in primary bladder tumors.

The ABO(H) histoblood group genes have been mapped by linkage analysis to 9q34.1-34.2, an area of common deletions in bladder cancer. Lack of ABO(H) antigen expression in bladder tumors is a frequent and well-documented event. In bladder neoplasms the loss of A and B transferase activity is due to down-regulation of the ABO gene transcripts. Our study was undertaken in order to determine the presence of structural alterations of the ABO(H) gene-encoding locus in primary bladder tumors, to estimate the extent of allelic deletions and to characterize further the pattern of histoblood group antigen expression. Fifty-three primary bladder tumors were analyzed by immuno-histochemistry (IHC) using a panel of well-characterized antibodies and fresh frozen tissue sections. Normal and tumor DNA also were analyzed by PCR coupled with restriction enzyme analysis in order to establish the ABO genotype. Results obtained from these analyses were then compared to allelotyping data at the 9q34.1-4 region by Southern blotting. IHC data showed undetectable levels of antigen expression on neoplastic cells in 59% of informative cases. PCR-based genotypic results revealed allelic losses in 27% of heterozygous cases. Four of the 16 pheno- and/or genotypically altered cases (25%) presented loss of heterozygosity at D9S10 or D9S7 loci. Our data indicate that the lack of ABO antigen expression in certain bladder tumors is due to the allelic loss of the ABO glycosyltransferase-encoding genes and that in some of these tumors the loss involves the surrounding chromosomal region at 9q34.1-4.

Female urethral carcinoma: an analysis of treatment outcome and a plea for a standardized management strategy.

OBJECTIVE: To evaluate our experience with primary carcinomas of the female urethra, by analysing the impact of tumour variables and treatment on overall, disease-specific, local recurrence- and metastasis-free survival. PATIENTS AND METHODS: Between 1958 and 1994, 72 women (median age 60 years, mean 59, range 21-84) with primary urethral carcinoma were identified. They were followed for a median (range) of 85 (0-384) months. The patients were stratified by stage, nodal status, histology, treatment, type of surgery, site of disease, year of diagnosis and smoking habit. RESULTS: In a univariate analysis, stage, nodal status, type of surgery and site of the disease were important factors for survival and recurrence. In a multivariate analysis, primary stage, nodal status and site of disease were independent predictors of survival. CONCLUSION: Current modalities of treatment are ineffective for local control and survival; new treatment strategies are needed for female urethral cancers.

Cigarette smoking and chromosome 9 alterations in bladder cancer.

Epidemiological studies suggest that bladder cancer may be caused by carcinogens in tobacco and certain occupational exposures. Molecular studies have shown that chromosome 9 alterations and TP53 mutations are the most frequent events in bladder cancer. To date, the relationships between epidemiological risk factors and genetic alterations have not been fully explored in bladder cancer. The purpose of this study was to explore the association between smoking and chromosome 9 aberrations in bladder cancer cases. Seventy-three patients with bladder cancer at Memorial Sloan-Kettering Cancer Center were evaluated for smoking history, occupational history, and chromosome 9 alterations. The epidemiological data were abstracted from medical charts. Patients' tumor tissues were analyzed using RFLP and microsatellite polymorphism assays for detection of chromosome 9 alterations. Elevated odds ratios (ORs) were found for chromosome 9 alterations in smokers compared to those in nonsmokers (OR = 4.2; 95% confidence interval, 1.02-17.0) after controlling for age, sex, race, occupational history, and stage of disease. The ORs were 3.6 for those smoking < or = 20 cigarettes per day and 5.8 for those smoking > 20 cigarettes per day. No association was found between occupational history and chromosome 9 alterations. This study supplies evidence suggestive of the link between smoking and chromosome 9 alterations in the etiology of bladder cancer and indicates that potential tumor suppressor genes on chromosome 9 may be involved in smoking-related bladder carcinogenesis.

[Clinical value of the study of proliferation and cell activation antigens with flow cytometry in bladder cancer]. / Intérêt clinique de l'étude d'antigènes de prolifération et d'activation cellulaire par cytométrie en flux dans le cancer de la vessie.

The proliferative rate of tumor cells is frequently proportional to their degree of aggressiveness. The objective of the present study was to determine the correlation between the expression of three antigens associated with proliferation and the stage and ploidy of bladder cancers. The reactivity of antibodies against the nuclear antigen Ki-67 and membrane antigens T43 and the epidermal growth factor receptor (EGFR) was studied by flow cytometry on a series of 35 clinical samples of superficial and infiltrating bladder cancer and as well as on 5 specimens of normal urothelial cells. A preferential expression of the T43 antigen was observed on invasive cancer. EGFR was less frequently expressed; however, seven of the nine positive samples were from invasive cancers. Ki67 on the other hand did not show any selective expression according to tumor stage. When comparing Ki-67 and T43 expression on individual tumor samples, a negative correlation was found in that no tumor strongly expressed both markers simultaneously. These results suggest that markers of proliferation and activation do not all measure the same tumor parameters. Together, they may provide prognostic information that may be useful in the follow-up of patients with bladder cancer.

Overexpression of p53 protein in a high-risk population of patients with superficial bladder cancer before and after bacillus Calmette-Guérin therapy: correlation to clinical outcome.

PURPOSE: We have previously demonstrated that p53 overexpression is predictive of disease progression and survival in Ta, Tis, and T1 tumors. Instillation of Bacillus Calmette-Guérin (BCG) is now accepted to be the most efficient adjuvant therapy for superficial bladder carcinoma. The aim of this study was to determine if p53 status, assessed before and after intravesical BCG therapy, can predict clinical outcome in a high-risk population of patients with superficial bladder carcinoma. MATERIALS AND METHODS: We examined 196 tissue specimens from 98 patients, obtained immediately before and after intravesical BCG therapy. The pretherapy population was composed of 22 Ta, 57 Tis, and 19 T1 tumors. After BCG, 66 specimens were TO and 32 had residual tumors. Nuclear p53 overexpression was analyzed in relation to time to disease progression and disease-specific survival. RESULTS: The median follow-up duration was 44 months. The detection of nuclear p53 overexpression before BCG therapy did not predict response to BCG therapy. Pre-BCG p53 protein overexpression, response to BCG therapy, and pre-BCG stage were all independent markers of disease progression. In patients with residual disease after BCG therapy (nonresponders), multivariate analysis confirmed that posttherapy p53 overexpression was the only independent marker of disease progression. CONCLUSION: In this high-risk population of patients with superficial bladder tumors, patients who have p53 nuclear overexpression in the tumor and stage T1 disease before BCG therapy are at high risk of disease progression. Furthermore, in the group of patients with residual disease after BCG therapy, p53 status is a better predictor of disease progression than post-BCG stage.

Microsatellite instability and deletion analysis of chromosome 10 in human prostate cancer.

Despite the clinical relevance of prostate cancer, few aspects regarding the molecular alterations involved in the process of prostate carcinogenesis are clearly understood. Cytogenetic and molecular genetic studies have identified specific abnormalities in prostate tumors, mainly on chromosomes 8, 10 and 16. On the basis of these findings, we designed a study to further characterize the altered regions on chromosome 10, using 15 microsatellite markers on a population composed of 20 paired normal and primary non-metastatic prostatic-tumor samples. Overall, 65% (13/20) of the cases analyzed showed molecular alterations, mainly rearrangements and deletions. The locus presenting the highest rate of abnormalities was D1OS221, which maps to 10q23-q24. Another region with frequent alterations was 10q21, at the DIOS109 locus. There was no statistical association between microsatellite abnormalities and Gleason grade or tumor stage in the prostate cancer cases studied. These results suggest that microsatellite alterations on the long arm of chromosome 10 are non-random events occurring in prostate cancer and that they may play a role in the process of tumorigenesis in these neoplasms.

Analysis of p21WAF1/CIP1 in primary bladder tumors.

The p21WAF1/CIP1 gene is regulated by p53 and encodes a cyclin-dependent kinase (Cdk)-inhibitor involved in senescence and cell quiescence. The role of p21 as a negative regulator of cell proliferation suggests that it may function as a tumor suppressor gene. However, only a few mutations of the p21WAF1/CIP1 gene have been reported to date. In order to assess potential p21WAF1/CIP1 gene alterations in human bladder cancer, we have examined this gene and its encoded product in a well-characterized cohort of 27 primary bladder tumors. Mobility shifts by single-strand conformation polymorphism in the p21WAF1/CIP1 gene were identified in 2 cases. Sequencing analyses revealed that one of these cases had point mutations in the 3' untranslated region, while the other case had a frame shift mutation at positions 322 (C to A) and a deletion of 8 nucleotides (323-->331; CCG-->ACG, codon 81 Arg-->Thr) that produced a stop signal at codon 83 (Gly--Stop). This tumor had a p21-negative phenotype by immunohistochemistry, but did not lose any allele. We further characterized these cases by the study of TP53 mutations using single-strand conformation polymorphism (PCR-SSCP) and sequencing, as well as immunohistochemical assays. Seven mobility shifts were identified and seven cases showed p53 nuclear accumulation. The two cases displaying mutated p21WAF1/CIP1 had wild-type TP53. It is concluded that p21WAF1/CIP1 gene aberrations are infrequent in bladder carcinoma but may be occasionally identified in primary bladder tumors.

Deletion of the p16 and p15 genes in human bladder tumors.

BACKGROUND: Two genes, p16 (also known as CDKN2, INK4A, or MTS1) and p15 (also described as INK4B or MTS2), are found in tandem at chromosome 9p21. These genes are designated as candidate tumor suppressor genes because they encode proteins that function as negative cell cycle regulators. (The encoded polypeptides inactivate specific cyclin-protein kinase complexes that are required for progression through the cell cycle.) Molecular genetic studies have revealed that deletion of the p16 and p15 genes occurs frequently in cancer cell lines and in certain malignant neoplasms. PURPOSE: We evaluated the frequency of p16 and p15 gene alterations in a well-characterized cohort of human transitional cell bladder cancers, and we explored potential associations between alterations in these genes and tumor stage and/or grade. METHODS: Tumor tissue and normal tissue from 110 patients with transitional cell carcinoma of the urinary bladder were examined. The status of the p16 and p15 genes in these tissues was determined by Southern blotting and hybridization with gene-specific probes, by coupled polymerase chain reaction and single-strand conformation polymorphism analysis (PCR-SSCP), and by sequencing DNA fragments produced during PCR. Associations between alterations in the genes and tumor stage and/or grade were evaluated using the two-tailed Fisher's exact test. RESULTS: Homozygous deletion (both alleles lost) of the p16 and the p15 genes was observed in 11 and nine bladder tumors, respectively. Eight of the 11 tumors exhibiting complete loss of the p16 gene also displayed homozygous deletion of the p15 gene. Exclusive loss of either gene was detected in only three tumors. Hemizygous deletion (one allele lost, also referred to as loss of heterozygosity [LOH] of the p16 and/or p15 genes was observed in eight tumors. Rearrangement of the two genes was indicated in three additional tumors. No point mutations were identified in either gene. The overall frequency of alteration in this cohort of bladder tumors was approximately 18% for each gene (in 20 [18.3%, 95% confidence interval (CI) = 11.1%-25.6%] of 109 informative tumors for the p16 gene and in 18 [18%, 95% CI = 10.5%-25.5%] of 100 informative tumors for the p15 gene). A statistically significant association between p16 gene alteration and bladder tumors of low stage (P < .01) and grade (P < .01) was observed; a significant association between p15 gene alteration and tumors of low stage (P < .01) was also detected. CONCLUSIONS: Alteration of the p16 and p15 genes, especially coincident homozygous deletion, appears to be a common event in bladder cancer.

Genetic evidence in melanoma and bladder cancers that p16 and p53 function in separate pathways of tumor suppression.

The 9p21 region of human chromosome 9 is a hot spot for chromosomal aberrations in both cultured cell lines and primary tumors. This region contains a gene, P16 (also called MTS1, CDKN2 and p16INK4), that encodes a presumptive negative cell cycle regulator called p16. P16 is deleted or mutated at high frequency in a variety of tumor cell lines including melanoma and bladder carcinoma lines. As such, it is likely to be a tumor suppressor gene. Here we show that P16 is mutated in primary bladder carcinomas (3 of 33) and melanomas (5 of 34). These findings support studies that show P16 mutations are not solely a product of growth in tissue culture but rather are involved in formation of tumors in viva. Some bladder primary tumors and some bladder and melanoma tumor cell lines contain mutations in both P16 and P53 at frequencies that suggest that p53 and p16 function in different pathways, each of which is important in suppressing malignant transformation.

p27Kip1: chromosomal mapping to 12p12-12p13.1 and absence of mutations in human tumors.

The p27Kip1 gene codes for a cyclin-dependent kinase inhibitor implicated in G1 arrest by transforming growth factor beta, cell-cell contact, agents that elevate cyclic AMP, and the growth-inhibitory drug rapamycin. p27 binds to and inhibits complexes formed by cyclin E-cdk2, cyclin A-cdk2, and cyclin D-cdk4. The involvement of p27 in the negative regulation of cell proliferation suggests that it may also function as a tumor suppressor gene. Using a combination of somatic cell hybrid panels and fluorescence in situ hybridization p27Kip1 has been mapped to the short arm of chromosome 12 at the 12p12-12p13.1 boundary, reported to harbor deletions and rearrangements in leukemia and mesotheliomas. In order to assess potential p27Kip1 gene alterations, we have screened a total of 147 human primary solid tumors and found no detectable cancer-specific mutations. These results argue that the often observed loss of antimitogenic transforming growth factor beta responsiveness in human cancer cells is not due to structural defects in p27Kip1.