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Structure and Methylation-Based Silencing of a Gene (DBCCR1) Within a Candidate Bladder Cancer Tumor Suppressor Region at 9q32-q33
PRINCIPLES OF ONCOLOGY, AND TUMORS OF BLADDER, PENIS AND URETHRA
regulation and iii) whether p21 can be induced by p53. WAFlIp21 mRNA levels examined in four cell lines were comparable to bladder mucosa. One cell line, HT1376, failed to express p21 protein due to a frame shift mutation. Overexpression of WAFlIp2 1 cDNA inhibited clone formation in three cell lines, whereas transfection with antisense WAFl increased clone sizes and numbers. WAFl sense clones showed diminished cell proliferation compared to the parental cell line. Apoptosis-induced wild-type p53 was not inhibited by overexpression of antisense WAFlIp21. In a cell clone derived from line VMCubl by stable transfection with wild-type p53 under the control of a metallothionein promoter. p21 was induced along with p53 upon activation of the promoter with zinc chloride. This induction was accompanied by a decrease in cell proliferation but by little apoptosis. These data suggest that p21 inhibits proliferation in a p53-dependent or independent manner but does not mediate p53-induced apoptosis in urothelial carcinoma cells.
Editorial Comment: Several genes that have achieved prominence in our understanding of the biology of urological cancer have been revealed to affect various aspects of the normal cell cycle. As a consequence of defects in their structure they may, therefore, have a role in the proliferative and progressive aspects of these malignancies. The subject of this article, the WAFYCIP1/SDI1 gene which encodes the p21 protein, has attracted attention because of its possible pivotal effect in normal cell regulation and cancer cell progression. The p21 protein product is an inhibitor of several cyclin-dependent kinases and can inhibit DNA replication. Through its interaction with proliferating cell nuclear antigens it is thought to mediate some of the effects of the tumor suppressor p53, particularly cell cycle arrest in the G1 phase in response to DNA damage, and may mediate p53 induced apoptosis. However, its relationship with p53 expression in normal and malignant urothelium remains unclear. For example, the promoter region of WAFllp21 has 2 p53 responsive elements through which p53 may induce transcription of p21. However, induction of p21 may also occur in a p53 independent manner, since it has been observed in normal tissues during development or differentiation in the absence of p53 activation. The authors examined the expression of WAFllp21 messenger ribonucleic acid in several bladder cancer cell lines, all of which contained mutated p53, and the effect on these cell lines of transfection of expression of WAFllp21. Overexpression of WAF'llpZl or down regulation of expression (by transfection of antisense constructs) suggested that WAFl/p21 functioned in a direct growth suppressive manner. Since all of the cell lines examined contained only mutated p53, the growth inhibitory effect of p21 was believed to be independent of p53. Immunohistochemical observations in other studies have suggested that tumors with undetectable p21 and inactivated p53 may have a worse prognosis than tumors with inactivation of either of these proteins alone, which is in keeping with the presumed independent functional aspect of these substances. These data suggest further that the induction of p21 may mediate cell cycle arrest rather than apoptosis as elicited by p53. Thus, while WAF'llp21 may be important in p53 mediated G1 arrest in urothelial cancer cell lines, it does not appear to be involved in p53 induced apoptosis. Elicitation of such distinctions in molecular pathways may be important in our understanding of the regulatory function of various genes. Based on our understanding of distinct biological pathways of different types of urothelial malignancies, this may permit selective therapeutic approaches in manipulating those pathways by stimulating or inhibiting the transcriptional expression of these genes. Michael J. Droller, M.D.
Structure and Methylation-Based Silencing of a Gene (DBCCR1) Within a Candidate Bladder Cancer Tumor Suppressor Region at 9q32-q33 T. HABUCHI, M. LUSCOMBE, P. A. ELDER AND M. A. KNOWLES, Molecular Genetics Laboratory, Marie Curie Research Institute, Surrey, United Kingdom Genomics, 4 8 277-288, 1998 Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, indicating the presence of one or more relevant tumor suppressor genes. We previously mapped one of these putative tumor suppressor loci to 9q32q33 and localized the candidate region within a single YAC 840 kb in size. This locus has been designated DBCl (for deleted in bladder cancer gene 1). We have identified a novel gene, DBCCR1, in t h i s candidate region by searching for expressed sequence tags (ESTs)that map to YACs spanning the region. Database searching using the entire DBCCRl cDNA sequence identified several human ESTs and a few homologous mouse ESTs. However, the predicted 761-amino-acid sequence had no sigdicant homology to known protein sequences. Mutation analysis of the coding region and Southern blot analysis detected neither somatic mutations nor gross genetic alterations in primary TCCs. Although DBCCRl was expressed in multiple normal human tissues including mthelium, mRNA expression was absent in 5 of 10 (50%)bladder cancer cell lines. Methylation analysis of the CpG island at the 5' region of
715
716
PRINCIPLES OF ONCOLOGY, AND TUMORS OF BLADDER, PENIS AND URETHRA
the gene and the induction of de nwo explxssion by a demethylathg agent indicated that this island might be a fresuent target for hypemethylation and that hypermethylation-based silencing of the gene occurs in TCC. These findings make DBCCRl a good candidate for DBC1. Editorial Comment: Loss of heterozygosity and deletion of chromosome a r m s 9q or Sp have been found in more than 5oo/o of superficial and invasive urothelial cancers. Because this finding has been more common in low grade papillary transitional cell cancer, however, this locus has been associated with simple cellular proliferation rather than invasiveness. Therefore, they may be responsible for the early phases in malignant tumor development although they have come to be associated with less aggressive forms of urothelial malignancy. The authors describe a new gene, DBCCRl, located within a critical region at 9q32 to q33, and report that its hypermethylation based loss of function is associated with an increased proliferative activity of tumor cells. Definition of this gene locus has been difficult but may well provide the key in our understanding as to how the cascade for tumor cell proliferation and carcinogenesisis initiated. Michael J. Droller, M.D.
Is Chromosome 9 Loss a Marker of Disease Recurrence in Transitional Cell Carcinoma of the Urinary Bladder?
J. M. S. BARTLETT, A. D. WATERS, S, A. BALLANTYNE, J. J. GOING,K. M. GRIGOR AND T. G. Coom, Departments of Surgery and Pathology, Glasgow University, Glasgow Royal Infirmary, Glasgow and Department of Pathology, Edinburgh University, Edinburgh, United Kingdom Brit. J. Cancer, 77: 2193-2198,1998 Investigation of transitional cell carcinoma of the urinary bladder (TCC) patients classified by recurrence andor progression has demonstrated that loss of chromosome 9, as detected by FISH analysis of the pericentromeric classical satellite marker at 9q12, occurs early. A total of 105 TCCs from 53 patients were analyzed in situ by two independent observers for loss of chromosome 9 using quantitative chromosomal copy number were defined for normal skin epidermis and bladder epithelium. Values for chromosome 9 copy number out with the range 1.51-2.10 (mean 2 3 x s.d. of normal values) were significantly abnormal. Twenty-five TCCs were detected with consistent monosomic scores. Of 89 TCCs, in which multiple tumour areas were analyzed, 85 tumours (96%) demonstrated the same chromosome 9 copy number in all areas (2-6) analyzed; only three tumours demonstrated heterogeneity for this locus. A total of 36% (12 out of 33) of patients with subsequent disease recurrence demonstrated loss of chromosome 9 in their primary and all subsequent TCCs analyzed. Only a single patient (n = 20) with non-recurrent TCC showed loss of chromosome 9 (P = 0.0085). Of 53 primary tumours, eight showed significant elevation of chromosome 17. Of these patients, six demonstrated elevation in chromosome 7 copy number. No abnormalities were observed in non-recurrent patients. This study describes rapid quantitation of chromosomal copy number by FISH using a pericentromeric probe for chromosome 9 in TCC of the urinary bladder. Routinely fured and processed material was evaluated without disaggregation. Strict quality control of FISH demonstrated that this technique was reproducible in a clinical environment and could be used to detect genetic changes relevant to patient outcome. It is proposed that loss of chromosome 9 from primary TCC of the urinary bladder identified patients at high risk of recurrence and possible progression. Editorial Comment: The authors examine the loss of a tumor suppressor gene at 9p21 and suggest that this may be an early event in urothelial carcinogenesis. Fluorescence in situ hybridization was used for probing archival bladder cancer material in 3 groups of patients, including those with a solitary episode of superficial bladder cancer and no subsequent disease recurrence, those with superficial urothelial cancers and repeated recurrences but without progression, and those with initially superficial papillary urothelial cancer and progression t o muscle invasion or metastases. Those patients with no recurrences demonstrated only 5% monosomy for chromosome 9, whereas those who had recurrent superficial cancer and progression had 36% monosomy at this locus. Thus, loss of this locus indicated increased risk for recurrent transitional cell carcinoma. The consistency that was found in this genetic alteration not only within a tumor sample that was taken at a single point in time, but also within recurrent tumors suggests that this marker may be useful in early identification of patients at risk for recurmnce. Of equal interest would be the role that this marker might have biologically in allowing proliferation of cells to occur and possibly functioning as an apparent trigger for subsequent events in the development of urothelial malignancies. Michael J. Droller, M.D.
regulation and iii) whether p21 can be induced by p53. WAFlIp21 mRNA levels examined in four cell lines were comparable to bladder mucosa. One cell line, HT1376, failed to express p21 protein due to a frame shift mutation. Overexpression of WAFlIp2 1 cDNA inhibited clone formation in three cell lines, whereas transfection with antisense WAFl increased clone sizes and numbers. WAFl sense clones showed diminished cell proliferation compared to the parental cell line. Apoptosis-induced wild-type p53 was not inhibited by overexpression of antisense WAFlIp21. In a cell clone derived from line VMCubl by stable transfection with wild-type p53 under the control of a metallothionein promoter. p21 was induced along with p53 upon activation of the promoter with zinc chloride. This induction was accompanied by a decrease in cell proliferation but by little apoptosis. These data suggest that p21 inhibits proliferation in a p53-dependent or independent manner but does not mediate p53-induced apoptosis in urothelial carcinoma cells.
Editorial Comment: Several genes that have achieved prominence in our understanding of the biology of urological cancer have been revealed to affect various aspects of the normal cell cycle. As a consequence of defects in their structure they may, therefore, have a role in the proliferative and progressive aspects of these malignancies. The subject of this article, the WAFYCIP1/SDI1 gene which encodes the p21 protein, has attracted attention because of its possible pivotal effect in normal cell regulation and cancer cell progression. The p21 protein product is an inhibitor of several cyclin-dependent kinases and can inhibit DNA replication. Through its interaction with proliferating cell nuclear antigens it is thought to mediate some of the effects of the tumor suppressor p53, particularly cell cycle arrest in the G1 phase in response to DNA damage, and may mediate p53 induced apoptosis. However, its relationship with p53 expression in normal and malignant urothelium remains unclear. For example, the promoter region of WAFllp21 has 2 p53 responsive elements through which p53 may induce transcription of p21. However, induction of p21 may also occur in a p53 independent manner, since it has been observed in normal tissues during development or differentiation in the absence of p53 activation. The authors examined the expression of WAFllp21 messenger ribonucleic acid in several bladder cancer cell lines, all of which contained mutated p53, and the effect on these cell lines of transfection of expression of WAFllp21. Overexpression of WAF'llpZl or down regulation of expression (by transfection of antisense constructs) suggested that WAFl/p21 functioned in a direct growth suppressive manner. Since all of the cell lines examined contained only mutated p53, the growth inhibitory effect of p21 was believed to be independent of p53. Immunohistochemical observations in other studies have suggested that tumors with undetectable p21 and inactivated p53 may have a worse prognosis than tumors with inactivation of either of these proteins alone, which is in keeping with the presumed independent functional aspect of these substances. These data suggest further that the induction of p21 may mediate cell cycle arrest rather than apoptosis as elicited by p53. Thus, while WAF'llp21 may be important in p53 mediated G1 arrest in urothelial cancer cell lines, it does not appear to be involved in p53 induced apoptosis. Elicitation of such distinctions in molecular pathways may be important in our understanding of the regulatory function of various genes. Based on our understanding of distinct biological pathways of different types of urothelial malignancies, this may permit selective therapeutic approaches in manipulating those pathways by stimulating or inhibiting the transcriptional expression of these genes. Michael J. Droller, M.D.
Structure and Methylation-Based Silencing of a Gene (DBCCR1) Within a Candidate Bladder Cancer Tumor Suppressor Region at 9q32-q33 T. HABUCHI, M. LUSCOMBE, P. A. ELDER AND M. A. KNOWLES, Molecular Genetics Laboratory, Marie Curie Research Institute, Surrey, United Kingdom Genomics, 4 8 277-288, 1998 Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, indicating the presence of one or more relevant tumor suppressor genes. We previously mapped one of these putative tumor suppressor loci to 9q32q33 and localized the candidate region within a single YAC 840 kb in size. This locus has been designated DBCl (for deleted in bladder cancer gene 1). We have identified a novel gene, DBCCR1, in t h i s candidate region by searching for expressed sequence tags (ESTs)that map to YACs spanning the region. Database searching using the entire DBCCRl cDNA sequence identified several human ESTs and a few homologous mouse ESTs. However, the predicted 761-amino-acid sequence had no sigdicant homology to known protein sequences. Mutation analysis of the coding region and Southern blot analysis detected neither somatic mutations nor gross genetic alterations in primary TCCs. Although DBCCRl was expressed in multiple normal human tissues including mthelium, mRNA expression was absent in 5 of 10 (50%)bladder cancer cell lines. Methylation analysis of the CpG island at the 5' region of
715
716
PRINCIPLES OF ONCOLOGY, AND TUMORS OF BLADDER, PENIS AND URETHRA
the gene and the induction of de nwo explxssion by a demethylathg agent indicated that this island might be a fresuent target for hypemethylation and that hypermethylation-based silencing of the gene occurs in TCC. These findings make DBCCRl a good candidate for DBC1. Editorial Comment: Loss of heterozygosity and deletion of chromosome a r m s 9q or Sp have been found in more than 5oo/o of superficial and invasive urothelial cancers. Because this finding has been more common in low grade papillary transitional cell cancer, however, this locus has been associated with simple cellular proliferation rather than invasiveness. Therefore, they may be responsible for the early phases in malignant tumor development although they have come to be associated with less aggressive forms of urothelial malignancy. The authors describe a new gene, DBCCRl, located within a critical region at 9q32 to q33, and report that its hypermethylation based loss of function is associated with an increased proliferative activity of tumor cells. Definition of this gene locus has been difficult but may well provide the key in our understanding as to how the cascade for tumor cell proliferation and carcinogenesisis initiated. Michael J. Droller, M.D.
Is Chromosome 9 Loss a Marker of Disease Recurrence in Transitional Cell Carcinoma of the Urinary Bladder?
J. M. S. BARTLETT, A. D. WATERS, S, A. BALLANTYNE, J. J. GOING,K. M. GRIGOR AND T. G. Coom, Departments of Surgery and Pathology, Glasgow University, Glasgow Royal Infirmary, Glasgow and Department of Pathology, Edinburgh University, Edinburgh, United Kingdom Brit. J. Cancer, 77: 2193-2198,1998 Investigation of transitional cell carcinoma of the urinary bladder (TCC) patients classified by recurrence andor progression has demonstrated that loss of chromosome 9, as detected by FISH analysis of the pericentromeric classical satellite marker at 9q12, occurs early. A total of 105 TCCs from 53 patients were analyzed in situ by two independent observers for loss of chromosome 9 using quantitative chromosomal copy number were defined for normal skin epidermis and bladder epithelium. Values for chromosome 9 copy number out with the range 1.51-2.10 (mean 2 3 x s.d. of normal values) were significantly abnormal. Twenty-five TCCs were detected with consistent monosomic scores. Of 89 TCCs, in which multiple tumour areas were analyzed, 85 tumours (96%) demonstrated the same chromosome 9 copy number in all areas (2-6) analyzed; only three tumours demonstrated heterogeneity for this locus. A total of 36% (12 out of 33) of patients with subsequent disease recurrence demonstrated loss of chromosome 9 in their primary and all subsequent TCCs analyzed. Only a single patient (n = 20) with non-recurrent TCC showed loss of chromosome 9 (P = 0.0085). Of 53 primary tumours, eight showed significant elevation of chromosome 17. Of these patients, six demonstrated elevation in chromosome 7 copy number. No abnormalities were observed in non-recurrent patients. This study describes rapid quantitation of chromosomal copy number by FISH using a pericentromeric probe for chromosome 9 in TCC of the urinary bladder. Routinely fured and processed material was evaluated without disaggregation. Strict quality control of FISH demonstrated that this technique was reproducible in a clinical environment and could be used to detect genetic changes relevant to patient outcome. It is proposed that loss of chromosome 9 from primary TCC of the urinary bladder identified patients at high risk of recurrence and possible progression. Editorial Comment: The authors examine the loss of a tumor suppressor gene at 9p21 and suggest that this may be an early event in urothelial carcinogenesis. Fluorescence in situ hybridization was used for probing archival bladder cancer material in 3 groups of patients, including those with a solitary episode of superficial bladder cancer and no subsequent disease recurrence, those with superficial urothelial cancers and repeated recurrences but without progression, and those with initially superficial papillary urothelial cancer and progression t o muscle invasion or metastases. Those patients with no recurrences demonstrated only 5% monosomy for chromosome 9, whereas those who had recurrent superficial cancer and progression had 36% monosomy at this locus. Thus, loss of this locus indicated increased risk for recurrent transitional cell carcinoma. The consistency that was found in this genetic alteration not only within a tumor sample that was taken at a single point in time, but also within recurrent tumors suggests that this marker may be useful in early identification of patients at risk for recurmnce. Of equal interest would be the role that this marker might have biologically in allowing proliferation of cells to occur and possibly functioning as an apparent trigger for subsequent events in the development of urothelial malignancies. Michael J. Droller, M.D.