High frequency of chromosome 9p allelic loss and CDKN2 tumor suppressor gene alterations in squamous cell carcinoma of the bladder.

BACKGROUND: In the Western Hemisphere, 90% of bladder cancers are transitional cell carcinomas, while only 7% are classified as squamous cell carcinomas. In contrast, in Egypt and regions of the Middle East and Africa, where infection by the trematode Schistosoma haematobium is endemic, squamous cell carcinoma is the most common bladder cancer as well as the most common cancer in men. PURPOSE: We planned experiments to understand the genetic defects underlying the development of squamous cell carcinoma and to determine if the morphologically and clinically distinct squamous cell carcinoma and transitional cell carcinoma of the bladder evolve following different genetic alterations. METHODS: Squamous cell carcinoma specimens from high-risk (Egypt, n = 19) and low-risk (Sweden, n = 12) populations were examined for genetic defects known to be involved in transitional cell carcinoma tumorigenesis. Homozygous deletions of the CDKN2 tumor suppressor gene were detected by comparative multiplex polymerase chain reaction. Mutations in the CDKN2 and p53 (also known as TP53) genes were analyzed by single-strand conformation polymorphism and DNA sequencing. Immunohistochemical staining of p53 protein was also performed. Allelic losses in chromosome arms 9p, 9q, and 17p were determined by microsatellite analysis. RESULTS: Homozygous deletions and sequence mutations in the CDKN2 gene were found in 67% (eight of 12) of squamous cell carcinoma specimens, a frequency three times higher than that reported for uncultured transitional cell carcinomas (P = .009). Hemizygous and homozygous deletions in 9p, where CDKN2 resides, were found in 92% (11 of 12) of uncultured squamous cell carcinomas, while only about 39% (35 of 90) of transitional cell carcinomas showed these losses (P = .001). Deletions in 9p with no change in 9q were found in 92% (10 of 11) of squamous cell carcinomas compared with only 10% (11 of 110) of transitional cell carcinomas (P < .001) reported in the literature. The frequency of p53 mutations in squamous cell carcinomas was similar to that reported for invasive transitional cell carcinomas (60%), but the type and position of mutations differed between the two tumor types. Allelic losses in chromosome arm 17p, where the p53 gene resides, were found to be less frequent in squamous cell carcinomas (38%) than in invasive transitional cell carcinomas (60%). CONCLUSIONS: Our results suggest that a putative tumor suppressor gene on 9p, possibly CDKN2, may contribute to squamous cell carcinoma tumorigenesis. Our data on squamous cell carcinoma and previously reported data on transitional cell carcinoma indicate that these two bladder carcinomas differ in their genetic alterations, suggesting that distinct underlying genetic defects may explain, at least in part, the pathological differences between the two tumors of the bladder epithelium. IMPLICATIONS: Development of diagnostic and therapeutic strategies for squamous cell carcinoma of the bladder based on its distinct genetic alterations is warranted.

Microsatellite instability in bladder cancer.

Somatic instability at microsatellite repeats was detected in 6 of 200 transitional cell carcinomas of the bladder. Instabilities were apparent as changes in (GT)n repeat lengths on human chromosome 9 for four tumors and as alterations in a (CAG)n repeat in the androgen receptor gene on the X chromosome for three tumors. Single locus alterations were detected in three tumors, while three other tumors revealed changes in two or more loci. In one tumor we found microsatellite instability in all five loci analyzed on chromosome 9. The alterations detected were either minor 2-base pair changes or larger (> 2 base pairs) alterations in repeat length. All six tumors were low stage (Ta-T1), suggesting that these alterations can occur early in bladder tumorigenesis.

Two molecular pathways to transitional cell carcinoma of the bladder.

Noninvasive transitional cell carcinomas of the bladder can have two distinct morphologies suggesting they contain different genetic alterations. Papillary transitional cell carcinomas (T(a) tumors) are often multifocal and only occasionally progress, whereas flat tumors (carcinomas in situ, CIS), frequently progress to invasive disease. We examined 216 bladder tumors of various stages and histopathologies for two genetic alterations previously described to be of importance in bladder tumorigenesis. Loss of heterozygosity of chromosome 9 was observed in 24 of 70 (34%) T(a) tumors but was present in only 3 of 24 (12%) CIS and dysplasia lesions (P = 0.04). In contrast, only 1 of 36 (3%) T(a) tumors contained a p53 gene mutation compared to 15 of 23 (65%) CIS and dysplasias (P < 0.001), a frequency comparable to that observed in muscle invasive tumors (25 of 49; 51%). The presence of p53 mutations in CIS and dysplasia could explain their propensities to progress since these mutations are known to destabilize the genome. Analysis of several tumor pairs involving a CIS and an invasive cancer provided evidence that the chromosome 9 alteration may in some cases be involved in the progression of CIS to more invasive tumors, in addition to its role in the initiation of T(a) tumors. However, the CIS and secondary tumor were found to contain different genetic alterations in some patients suggesting divergent progression pathways. Bladder carcinogenesis may therefore proceed through two distinct genetic alteration pathways responsible for generating superficial tumors with differing morphologies and pathologies.

Methylation of the 5' CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing.

Loss of heterozygosity on 9p21, where the p16/CDKN2 tumor suppressor and the p15INK4B cell cycle regulator genes are located, is a common genetic alteration in bladder cancer. However, it has been difficult to demonstrate homozygous deletions and intragenic mutations in either of these two genes in primary transitional cell carcinomas (TCC) of the bladder. Similarly, colon cancer-derived cell lines have shown no homozygous deletions of the p16/CDKN2 locus in contrast to a wide variety of tumor-derived cell lines. We have investigated abnormal methylation of the 5' CpG islands of the p16/CDKN2 and p15INK4B genes as an alternative mechanism of inactivation of these genes in bladder and colon cancers. De novo methylation of the 5' CpG island of p16/CDKN2 was observed in 12 of 18 (67%) uncultured bladder TCCs and in 2 of 3 (67%) bladder cell lines. In contrast, only 1 of 10 (10%) colon carcinomas showed methylation of the 5' CpG island of p16/CDKN2. It was striking to find that this region was extensively methylated and the gene not expressed in the normal colonic mucosa of 6 of 10 (60%) patients with colon cancer, whereas 5 of the corresponding colon tumors showed no methylation and high levels of p16/CDKN2 expression. Our data show a significant correlation (P = 0.00001, two-sided) between the absence of p16/CDKN2 expression and methylation of its 5' CpG island in bladder tumors, cell lines, and normal colon mucosa. In contrast, no association was observed between expression and methylation status of the 5' CpG island of p15INK4B. Our results suggest that the p16/CDKN2 tumor suppressor gene may be inactivated by methylation of its 5' CpG island in TCCs of the bladder. We also present evidence of methylation of the 5' CpG island in this autosomal gene in normal colonic tissue.

Evidence for two tumor suppressor loci associated with proximal chromosome 9p to q and distal chromosome 9q in bladder cancer and the initial screening for GAS1 and PTC mutations.

The most common genetic alteration identified to date in bladder cancer is loss of heterozygosity (LOH) of chromosome 9, suggesting the presence of possible tumor suppressor genes on this chromosome. We attempted to map the location of these genes by analyzing 69 primary transitional cell carcinomas of the bladder with a panel of microsatellite markers for LOH on chromosome 9. Monosomy 9 (defined by LOH of all informative markers analyzed on 9p and 9q) was detected in 26 of 69 (38%) tumors, and 22 of 69 (32%) tumors showed subchromosomal deletions. Twelve tumors (17%) demonstrated partial LOH of chromosome 9 and indicated two distinct regions of LOH. Eight tumors showed distal allelic loss of 9q with a minimal region of common deletion flanked proximally by marker GSN on 9q33. Six tumors showed proximal allelic loss of 9p and 9q with a minimal area of common deletion flanked by markers D9S970 on 9p12 and D9S283 on 9q21. Two tumors showed loss of both the distal region of 9q and the proximal region of 9p and 9q, which were separated by a possible 6-44 cM of retained genetic material. The proximal minimal area of common deletion excluded 9q22.3-q31 to where two putative tumor suppressor genes, the nevoid basal cell carcinoma syndrome and multiple self-healing squamous epithelioma (ESS1) genes, have been mapped. The growth arrest-specific gene (GAS1), a candidate tumor suppressor gene, was included within the proximal minimal region. We evaluated the GAS1 gene for its potential role in bladder cancer using single-strand conformational polymorphism to screen for mutations in GAS1 in 10 bladder cancer cell lines and 14 primary bladder tumors. A polymorphism at codon 88 was noted in one primary bladder tumor, but no other abnormalities were found, suggesting that another potential tumor suppressor gene important to bladder cancer resides in these minimally deleted regions. Because the nevoid basal cell carcinoma syndrome gene has long been speculated to be a putative tumor suppressor gene in bladder cancer and this gene has recently been characterized as the human homologue of the Drosophila patched gene (PTC), 20 primary bladder tumors with chromosome 9q LOH were screened for mutations in PTC using single-strand conformational polymorphism and heteroduplex analysis. No alterations were found in any of the samples analyzed. Furthermore, 4 of 37 noninvasive papillary (Ta) tumors demonstrated loss of all 9q markers with retention of 9p, whereas no Ta tumor showed loss of 9p with retention of all 9q markers, suggesting that LOH of 9q is the earlier event in bladder tumorigenesis. In summary, our results indicate two tumor suppressor loci associated with proximal chromosome 9p to q and distal chromosome 9q that may be important in bladder cancer. GAS1 and PTC do not seem to be frequently mutated in bladder cancer.

Role of chromosome 9 in human bladder cancer.

The tumors of 20 patients with multifocal primary transitional cell carcinoma of the bladder or lymph node metastases were examined for molecular genetic defects which we have previously found to be present in > 50% of invasive tumors. These included loss of heterozygosity (LOH) of chromosome 9, which occurs in superficial as well as invasive bladder tumors, and LOH of chromosome 17p and p53 mutations, which are commonly found only in invasive tumors. Analysis of multiple or recurrent primary tumors in 7 patients for these markers was generally consistent with recently published data that the tumors are monoclonal in origin and that p53 mutations occur as a late event in the generation of invasive bladder cancers. Comparison of the primary tumors and metastases to regional lymph nodes in 14 patients demonstrated a complete concordance between the molecular genetic defects present, showing that LOH of chromosomes 9 and 17p and p53 mutations occurred in the primary tumors before metastasis. Because of the importance of chromosome 9 in bladder cancer, we mapped the location of a putative tumor suppressor gene by restriction fragment length polymorphism analysis of 123 cases obtained in this and earlier studies. Most of the tumors showed LOH for more than one marker on chromosome 9. Results of mapping of 4 tumors with partial deletion of chromosome 9 suggests that the tumor suppressor gene is located between 9p12 and 9q34.1.

Absence of p53 gene mutations in primary nasopharyngeal carcinomas.

Alterations in the p53 tumor suppressor gene and Epstein-Barr virus status were investigated in 15 nasopharyngeal carcinoma (NPC) biopsies, 4 xenografts, and 2 cell lines from the Cantonese region of southern China. One other established NPC cell line obtained from a northern Chinese patient was also studied. Restriction fragment length polymorphism analysis revealed a loss of heterozygosity for chromosome 17p, where the p53 gene resides, in only one of 15 NPC biopsies. Polymerase chain reaction-single-stranded conformational polymorphism analysis and direct sequencing failed to detect sequence alterations in exons 5 through 8 of the p53 gene in the 15 tumors and in the 4 NPC xenografts, all of which tested positive for Epstein-Barr virus. In contrast, the 3 NPC cell lines were all negative for Epstein-Barr virus and contained G----C transversions in the p53 gene, with cell lines CNE-1 and CNE-2 harboring identical AGA (arginine) to ACA (threonine) changes at codon 280. These results suggest that p53 inactivation is not a necessary component of nasopharyngeal carcinogenesis in Cantonese but may be important in the establishment of cell lines derived from these tumors.

Presence of p53 mutations in primary nasopharyngeal carcinoma (NPC) in non-Asians of Los Angeles, California, a low-risk population for NPC.

Mutatins of the p53 tumor suppressor gene are rare in nasopharyngeal carcinoma (NPC) patients who reside in high-risk areas, such as Southeastern China. Among this high-risk group, a pre-existing infection with the EBV and consumption of Cantonese salted fish are closely associated with NPC. We investigated the prevalence of p53 mutations in 28 primary NPC specimens from white (including Hispanic) and African-American patients in Los Angeles, who are at low risk for NPC. Using PCR-based single-strand conformational polymorphism and direct sequencing, we found four mutations (14%) in exons 5-8 of the p53 gene in four patients. All were C-to-T transition mutations: two were present in exon 5-one at codon 142 [CCT (Pro)-->CTT (Leu)] and another at codon 144 [CAG (Gln)-->TAG (stop codon)]. The other two mutations were identified in exon 8: one at codon 273 [CGT (Arg)-->CAT (His)], a CpG site, and one at codon 271, a silent mutation [GAG (Glu)-->GAA (Glu)]. This is the first report investigating the presence of p53 missense mutations in NPC among a low-risk population. Our data indicate that p53 is also an infrequent event among NPC patients at low risk for the disease.

Chronic lithium treatment antagonizes glutamate-induced decrease of phosphorylated CREB in neurons via reducing protein phosphatase 1 and increasing MEK activities.

The cyclic AMP response element binding protein (CREB) has major roles in mediating adaptive responses at glutamatergic synapses and in the neuroprotective effects of neurotrophins. CREB has been implicated as a potential mediator of antidepressant actions. In vitro, chronic lithium treatment has been shown to promote neuronal cell survival. In the present study, we have used cultures of cerebellar granule neurons to analyze the effects of acute and chronic lithium treatment on the response to toxic concentrations of glutamate. Such concentrations of glutamate decrease the phosphorylation of CREB at serine(133) in an N-methyl-D-aspartate (NMDA) receptor-dependent manner. Chronic, but not acute, lithium treatment suppresses glutamate-induced decreases in phosphorylated CREB, and transfection studies indicate that chronic lithium, in the presence of a glutamate stimulus, markedly increases CRE-driven gene expression. Experiments with selected pharmacological reagents indicate that the glutamate-induced decreases in phosphorylated CREB are regulated primarily by protein phosphatase 1. Chronic lithium treatment not only decreases protein phosphatase 1 activity under these circumstances, but also augments glutamate-induced increases in MEK activity. PD 98059, a MEK inhibitor, prevents chronic lithium treatment from increasing phosphorylated CREB levels in glutamate-treated neurons. We conclude from these results that chronic lithium treatment is permissive for maintaining higher phosphorylated CREB levels in the presence of glutamate in part by decreasing protein phosphatase 1 activity and in part by increasing MEK activity. Higher levels of phosphorylated CREB and CRE-responsive genes such as bcl-2 may be responsible for lithium's reported effects on neuronal survival.

Regulation of neuronal nitric oxide synthase and identification of novel nitric oxide signaling pathways.

Neuronal nitric oxide synthase (nNOS) participate in a variety of physiologic and pathologic processes in the nervous system. nNOS was originally felt to be a constitutively expressed enzyme, but recent observations suggest that its levels are dynamically controlled in response to neuronal development, plasticity and injury. nNOS expression is regulated through alternative promoter usage through alternative mRNA splicing and it is likely that this plays an important role in the inducibility of gene expression in response to extracellular stimuli. Emerging data also suggests that NO may be the key mediator linking activity to gene expression and long-lasting neuronal responses through NO activating p21Ras through redox-sensitive modulation.

Specific interference with gene expression and gene function mediated by long dsRNA in neural cells.

Double-stranded (ds) RNA-induced sequence-specific interference with gene expression, RNA interference (RNAi), has been extensively used in invertebrates, allowing for efficient and high-throughput gene silencing and gene function analysis. In vertebrates, however, use of RNAi to study gene function has been limited due to non-specific effects induced by double-stranded RNA (dsRNA)-dependent protein kinase and interferon activation. dsRNA-induced specific inhibition of vertebrate gene expression has only been shown in embryonic and non-differentiated mammalian cells. In this report, we demonstrate dsRNA-induced specific interference of gene expression and gene function in partially as well as fully differentiated mouse neuroblastoma cells. Specific silencing was observed in the expression of an integrated transgene coding for green fluorescent protein and a variety of endogenous genes. Moreover, we show that RNAi-mediated inhibition of poly (ADP-ribose) polymerase (PARP) expression induced cellular resistance to oxygen-glucose deprivation, consistent with the role of PARP in ischemia-induced brain damage. Our results indicate that RNAi can be used as a powerful tool to study gene function in neural cells.

Histochemical analysis of nitric oxide synthase by NADPH diaphorase staining.

The presence of NOS coincides with that of NADPH diaphorase activity; therefore, NADPH diaphorase staining is widely used to detect NOS-containing cells in neural and non-neural tissues. With the appropriate fixation procedure, this method is capable of detecting cells containing any of the NOS isoforms.

Nitric oxide mediates N-methyl-D-aspartate receptor-induced activation of p21ras.

N-methyl-D-aspartate (NMDA) glutamate receptor-mediated increases in intracellular calcium are thought to play a critical role in synaptic plasticity. The mechanisms by which changes in cytoplasmic calcium transmit the glutamate signal to the nucleus, which is ultimately important for long-lasting neuronal responses, are poorly understood. We show that NMDA receptor stimulation leads to activation of p21(ras) (Ras) through generation of nitric oxide (NO) via neuronal NO synthase. The competitive NO synthase inhibitor, L-nitroarginine methyl ester, prevents Ras activation elicited by NMDA and this effect is competitively reversed by the NO synthase substrate, L-arginine. NMDA receptor stimulation fails to activate Ras in neuronal cultures from mice lacking neuronal NO synthase. NMDA-induced Ras activation occurs through a cGMP-independent pathway as 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a potent and selective inhibitor of guanylyl cyclase, has no effect on NMDA receptor-induced activation of Ras, and the cell-permeable cGMP analog, 8Br-cGMP, does not activate Ras. Furthermore, NO directly activates immunoprecipitated Ras from neurons. NMDA also elicits tyrosine phosphorylation of extracellular signal-regulated kinases, a downstream effector pathway of Ras, through a NO/non-cGMP dependent mechanism, thus supporting the physiologic relevance of endogenous NO regulation of Ras. These results suggest that Ras is a physiologic target of endogenously produced NO and indicates a signaling pathway for NMDA receptor activation that may be important for long-lasting neuronal responses.

Reduction of functional N-methyl-D-aspartate receptors in neurons by RNase P-mediated cleavage of the NR1 mRNA.

One approach to studying the functional role of individual NMDA receptor subunits involves the reduction in the abundance of the protein subunit in neurons. We have pursued a strategy to achieve this goal that involves the use of a small guide RNA which can lead to the destruction of the mRNA for a specific receptor subunit. We designed a small RNA molecule, termed 'external guide sequence' (EGS), which binds to the NR1 mRNA and directs the endonuclease RNase P to cleave the target message. This EGS has exquisite specificity and directed the RNase P-dependent cleavage at the targeted location within the NR1 mRNA. To improve the efficiency of this EGS, an in vitro evolution strategy was employed which led to a second generation EGS that was 10 times more potent than the parent molecule. We constructed an expression cassette by flanking the EGS with self-cleaving ribozymes and this permitted generation of the specified EGS RNA sequence from any promoter. Using a recombinant Herpes simplex virus (HSV), we expressed the EGS in neurons and showed the potency of the EGS to reduce NR1 protein within neurons. In an excitotoxicity assay, we showed that expression of the EGS in cortical neurons is neuroprotective. Our results demonstrate the utility of EGSs to reduce the expression of any gene (and potentially any splice variant) in neurons.

Dynamic regulation of neuronal NO synthase transcription by calcium influx through a CREB family transcription factor-dependent mechanism.

Neuronal nitric oxide (NO) synthase (nNOS) is dynamically regulated in response to a variety of physiologic and pathologic stimuli. Although the dynamic regulation of nNOS is well established, the molecular mechanisms by which such diverse stimuli regulate nNOS expression have not yet been identified. We describe experiments demonstrating that Ca(2+) entry through voltage-sensitive Ca(2+) channels regulates nNOS expression through alternate promoter usage in cortical neurons and that nNOS exon 2 contains the regulatory sequences that respond to Ca(2+). Deletion and mutational analysis of the nNOS exon 2 promoter reveals two critical cAMP/Ca(2+) response elements (CREs) that are immediately upstream of the transcription start site. CREB binds to the CREs within the nNOS gene. Mutation of the nNOS CREs as well as blockade of CREB function results in a dramatic loss of nNOS transcription. These findings suggest that nNOS is a Ca(2+)-regulated gene through the interactions of CREB on the CREs within the nNOS exon 2 promoter and that these interactions are likely to be centrally involved in the regulation of nNOS in response to neuronal injury and activity-dependent plasticity.

Requirement for nitric oxide activation of p21(ras)/extracellular regulated kinase in neuronal ischemic preconditioning.

The mechanisms underlying neuronal ischemic preconditioning, a phenomenon in which brief episodes of ischemia protect against the lethal effects of subsequent periods of prolonged ischemia, are poorly understood. Ischemia can be modeled in vitro by oxygen-glucose deprivation (OGD). We report here that OGD preconditioning induces p21(ras) (Ras) activation in an N-methyl-D-aspartate receptor- and NO-dependent, but cGMP-independent, manner. We demonstrate that Ras activity is necessary and sufficient for OGD tolerance in neurons. Pharmacological inhibition of Ras, as well as a dominant negative mutant Ras, block OGD preconditioning whereas a constitutively active form of Ras promotes neuroprotection against lethal OGD insults. In contrast, the activity of phosphatidyl inositol 3-kinase is not required for OGD preconditioning because inhibition of phosphatidyl inositol 3-kinase with a chemical inhibitor or with a dominant negative mutant does not have any effect on the development of OGD tolerance. Furthermore, using recombinant adenoviruses and pharmacological inhibitors, we show that downstream of Ras the extracellular regulated kinase cascade is required for OGD preconditioning. Our observations indicate that activation of the Ras/extracellular regulated kinase cascade by NO is a critical mechanism for the development of OGD tolerance in cortical neurons, which may also play an important role in ischemic preconditioning in vivo.

Manganese superoxide dismutase protects nNOS neurons from NMDA and nitric oxide-mediated neurotoxicity.

Neuronal nitric oxide synthase (nNOS) neurons kill adjacent neurons through the action of NMDA-glutamate receptor activation, although they remain relatively resistant to the toxic effects of NMDA and NO. The molecular basis of the resistance of nNOS neurons to toxic insults is unknown. To begin to understand the molecular mechanisms of the resistance of nNOS neurons, we developed a pheochromacytoma-derived cell line (PC12) that is resistant to the toxic effects of NO. We found through serial analysis of gene expression (SAGE) that manganese superoxide dismutase (MnSOD) is enriched in the NO-resistant PC12 cell-derived line (PC12-R). Antisense MnSOD renders PC12-R cells sensitive to NO toxicity and increases the sensitivity to NO in the parental, NO-sensitive PC12 line (PC12-S). Adenoviral transfer of MnSOD protects PC12-S cells against NO toxicity. We extended these studies to cortical cultures and showed that MnSOD is enriched in nNOS neurons and that antisense MnSOD renders nNOS neurons susceptible to NMDA neurotoxicity, although it has little effect on the overall susceptibility of cortical neurons to NMDA toxicity. Overexpression of MnSOD provides dramatic protection against NMDA and NO toxicity in cortical cultures, but not against kainate or AMPA neurotoxicity. Furthermore, nNOS neurons from MnSOD -/- mice are markedly sensitive to NMDA toxicity. Adenoviral transfer of MnSOD to MnSOD-/- cultures restores resistance of nNOS neurons to NMDA toxicity. Thus, MnSOD is a major protective protein that appears to be essential for the resistance of nNOS neurons in cortical cultures to NMDA mediated neurotoxicity.