Impaired alpha-interferon signaling in transitional cell carcinoma: lack of p48 expression in 5637 cells

The limited success of IFN-alpha therapy for clinical treatment of transitional cell carcinoma (TCC) has prompted us to investigate the responsiveness of TCC lines to IFN-alpha. The response to IFN-alpha in terms of 561 gene induction, an IFN-stimulated response element-containing IFN-alpha/beta-inducible gene, and IFN-stimulated gene factor 3 (ISGF3) formation was normal in primary human urothelial cells. We tested the antiproliferative effects of IFN-alpha in three TCC lines as a measure of IFN-alpha responsiveness, and variable patterns of growth inhibition were observed in three TCC lines. More than 90% growth inhibition was noted in TCCSUP cells, whereas only 40% and 10% inhibition by IFN-alpha was observed in 5637 and HT1197 cells, respectively. IFN-alpha treatment formed extremely low levels of ISGF3 in electrophoretic mobility shift assays in these later two relatively insensitive cells. In addition, expression of the 561 gene was significantly reduced in these two TCC lines by Northern blots. We have further identified a low expression level of Tyk2 in HT1197 cells compared with two other TCCs. This suggests that an extremely low ISGF3 level after IFN-alpha treatment may be due to low Tyk2 expression or other unidentified defects. In 5637 cells, p48 protein expression was undetectable. This undetectable p48 expression is not due to a deletion in the coding region because the correct size protein is detected following IFN-gamma treatment. Consequently, the ISGF3 complex formation and 561 gene induction were restored by IFN-gamma pretreatment plus IFN-alpha treatment. Introduction of p48 expressing plasmid into 5637 cells was sufficient to form the ISGF3 complex by IFN-alpha treatment, suggesting the defect lies in the expression of p48 protein in 5637 cells. Detailed mechanistic understanding of the action of IFNs in bladder cancer cell lines may explain the abrogated therapeutic response of IFN-alpha in the clinical treatment of TCCs.     

N-myc modulates expression of p73 in neuroblastoma

The human p73 gene is a homolog of p53, which has been localized to chromosome 1p36 in a region that is frequently deleted in neuroblastoma. Transfection of the p73 gene into neuroblastoma cells that lack detectable p73 protein has been shown to result in growth suppression and to induce neuronal differentiation. In this study, we have identified by means of restriction landmark genome scanning (RLGS) a genomic fragment that was frequently reduced in intensity in neuroblastomas. The cloned fragment contained exon 1 of p73 as well as intronic and promoter sequences. We investigated the genomic and expression status of p73 and N-myc in 34 neuroblastoma tumors and 12 neuroblastoma cell lines. Approximately a third of neuroblastomas in our series exhibited deletion of p73. Most tumors analyzed exhibited reduced expression of p73, as determined by quantitative RT-PCR, in the absence of detectable p73 gene deletion. The reduced expression of p73 correlated with overexpression of N-myc in a statistically significant manner. The N-myc gene was transfected into two neuroblastoma cell lines that lacked N-myc amplification to determine its effect on p73 RNA levels. p73 was detectable at low level by RT-PCR in untransfected SK-N-AS cells and became undetectable following N-myc transfection, whereas in SH-EP1 cells, p73 levels were substantially reduced following transfection but remained detectable. Our data suggest that the N-myc gene modulates expression of p73, allowing neuroblastoma cells to escape the growth suppressing properties of p73.     

Overexpression of the wild type p73 gene in human bladder cancer

p73, a first p53 relative, was recently identified and shown to be monoallelically expressed in a number of different human tissues. To determine the potential role of this gene in human bladder cancer, we investigated p73 expression levels, allelic expression patterns, and analysed p73 mutations in 23 unselected primary invasive bladder cancers with matched normal tissues and in seven bladder cancer cell lines. In a comparison between normal and tumor tissues using quantitative RT-PCR analysis, we found that p73 was overexpressed in 22/23 bladder cancers, sometimes as great as 20-fold. Allelic expression analysis using a C/T polymorphism in exon 2 and a newly identified T/C polymorphism in exon 5 revealed that p73 was biallelically expressed in both normal bladder and cancer tissues, suggesting that p73 is not imprinted in bladder tissue. Mutation screening of the p73 gene in bladder cancer DNAs using denaturing high-performance liquid chromatography analysis and DNA sequencing revealed no tumor-specific mutations in any coding exons of the p73 gene. These data suggest that the p73 is unlikely to be a tumor suppressor gene, but that overexpression of p73 may contribute to tumorigenesis in bladder cancer.     

Tumor-specific p73 up-regulation mediates p63 dependence in squamous cell carcinoma

p63 is essential for normal epithelial development and is overexpressed in the vast majority of squamous cell carcinomas (SCC). Recent work had shown that DeltaNp63alpha is essential for survival of SCC cells, raising the possibility that the p63 pathway may be an attractive therapeutic target in these tumors. Nevertheless, it is unknown whether a therapeutic window exists for inhibiting p63 in tumor cells versus normal epithelia. Here, we show that SCC cells are uniquely dependent on DeltaNp63alpha for survival, unlike normal p63-expressing epithelial cells, and that dependence is mediated through tumor-specific up-regulation of the related protein p73. In normal primary human keratinocytes, we find that inhibition of endogenous p63 by RNA interference (RNAi) induces p21(CIP1) expression, inhibits cell cycle progression, and ultimately promotes cellular senescence. In contrast, p63 inhibition in SCC cells induces proapoptotic bcl-2 family members and rapidly triggers apoptosis. Expression of p73 is low in uncultured basal keratinocytes but is markedly up-regulated in both SCC cell lines and primary tumors in vivo. Whereas p21(CIP1) induction following loss of p63 in normal cells is independent of p53 and p73, both proapoptotic gene induction and cell death following p63 RNAi in tumor cells are p73 dependent. Finally, ectopic p73 expression in primary keratinocytes does not affect baseline cell proliferation but is sufficient to trigger cell death following loss of p63. Together, these findings define a specific molecular mechanism of p63 dependence through p73 up-regulation, and they provide a rationale for targeting the p63 pathway as a therapeutic strategy in SCCs.     

Synergistic decline in expressions of p73 and p21 with invasion in esophageal cancers

The significance of the p73 gene, a homologue of the p53 gene, in esophageal cancers is not fully understood. In order to clarify the role of p73 expression in esophageal cancers, p73 expression was immunohistochemically investigated in 106 surgically resected esophageal cancers and the results were compared with various clinicopathological factors. In normal esophageal epithelium, the expression of p73 was observed only in the nuclei of basal cells. In esophageal cancers, p73 immunoreactivity was observed in all intraepithelial lesions except one cancer, and was reduced with cancer invasion, to 78% and 64% at superficial invasion and deep invasion sites, respectively. However, p73 expression was not correlated with any other clinicopathological factor. The expressions of p53 and p21 were also investigated in esophageal cancer. To evaluate the status of the p53 gene mutation immunohistochemically, two monoclonal antibodies (DO7 and PAb240) were used. There seemed to be an inverse correlation between p73 expression and p53 mutation. Moreover, the expression of p21 was highly correlated with p73 expression irrespective of the p53 mutation status. In human esophageal cancers, p73 expression decreased with increasing degree of tumor invasion, and its decreased expression in local advanced tumor caused down-regulation of p21 expression, which might reflect tumor progression.     

Expression of p53 in urothelial cell cultures from tumour-bearing and tumour-free patients.

An explant culture technique was used to culture normal urothelium from patients with muscle-invasive bladder cancer (transitional cell carcinoma, TCC) (n = 11) and from non-tumour-bearing patients (n = 60). Cell cultures were examined for expression of p53 using the monoclonal antibody p53-240. There was a statistically significant increase in p53 expression in normal urothelial cell cultures from patients with TCC (P < 0.0005). Normal urothelial cultures from patients with TCC also showed more rapid proliferation in vitro when compared with non-tumour-bearing patients (P < 0.0005). A subgroup of non-tumour-bearing patients (n = 14) showed > 5% of cells expressing p53. p53 expression in this subgroup was found to correlate with cell proliferation in vitro (r2 = 0.766). None of these urothelial specimens was observed to express p53 when paraffin-embedded preparations were stained with p53-D07 antibody prior to culture. The rate of cellular proliferation in this subgroup did not differ from that of normal urothelium from TCC patients. Twenty-two paraffin-embedded, muscle-invasive TCC specimens were also evaluated for p53 expression using p53-D07. The expression of p53 in these tumours did not differ from that observed in normal urothelial cell cultures from patients with TCC (P = 0.26). This study identifies an overexpression of p53 in normal urothelial cells from patients with TCC and in proliferating cultures from a significant subgroup of patients without malignant disease. Increased p53 expression in normal cultured urothelial cells from patients with bladder cancer implies a global change in the mechanisms controlling urothelial cell division. This may represent an early step in the pathway to carcinogenesis.     

Differential expression of p73 splice variants and protein in benign and malignant ovarian tumours

The p73 gene encodes a protein with substantial structural and functional similarities to the tumour-suppressor p53. Alternative splicing of p73 mRNA leads to expression of 6 known RNA species and proteins (alpha, beta, gamma, delta, epsilon, zeta). We analysed the expression of these splice variants in ovarian adenocarcinoma by RT-PCR followed by detection of amplicons with the Southern technique and by immunoblot in 32 malignant and benign epithelial ovarian tumour specimens and 3 ovarian adenocarcinoma cell lines (A2780, 2008, OVCAR-3). p73alpha mRNA was expressed in all 17 ovarian cancer specimens, and 14 of 17 expressed at least 3 splice variants. In contrast, a different expression pattern was present in the ovarian adenomas: p73alpha was detected in 6 of 12 benign tumours, and only 1 adenoma expressed 3 splice variants. p73 protein was expressed in 9 of 16 ovarian cancer specimens, in all cell lines and in 1 of 3 borderline tumours. In contrast, none of 9 ovarian adenomas expressed detectable amounts of p73 protein. Expression of p73 mRNA and protein was not correlated with FIGO stage and histological grade, but we observed a significant correlation with over-expression of p53 protein. In summary, epithelial ovarian cancers express a more complex p73 isoform pattern and higher levels of p73 mRNA and protein than ovarian adenomas.     

Allelic expression of p73 in human ovarian cancers

Background: The p73 gene is structurally related to the tumor suppressor gene p53. The role of p73 in tumor development is still unclear and no data on ovarian cancer are so far available. For this reason we have analyzed, in a panel of ovarian cancers, the allelic distribution and expression of p73.Patients and methods: Fifty-one samples from ovarian cancers and five human ovarian cancer cell lines growing in culture were analyzed. Allelic origin was analyzed by PCR after digestion with the restriction enzyme Sty I. Heterozygous, informative cases were selected for studies aimed at evaluating allelic expression of p73.Results: We found an allelic distribution similar to that previously reported. LOH was found in two patients with ovarian cancer. In one case in which normal ovarian tissue was available biallelic expression of p73 was found.Conclusion: In comparisons of ovarian cancers and borderline tumors, no differences in allelic distribution and/or expression were found, suggesting that p73 does not play an important role in the pathogenesis and development of ovarian cancer.     

Activity of E2F-dependent promoters in bladder carcinoma cells and their use for tumour-specific targeting of p53-induced apoptosis

Inactivation of P53 and RB functions are crucial changes in bladder cancer (TCC). High-level re-expression of P53 elicits apoptosis in TCC cell lines, but also--as shown here--in normal uroepithelial cells. Compromised RB function is thought to cause increased activity of E2F-dependent promoters in carcinoma cells. Indeed, several, but not all E2F-dependent promoters were stronger in TCC lines than in normal cells, with the highest activities in cell lines lacking RB rather than p16INK4A. Re-expression of p53 from an E2F-dependent promoter suppressed clone formation and induced apoptosis in TCC lines as efficiently as expression from the stronger RSV-LTR or LINE-1 promoters. In normal cells, p53 expression from an E2F-dependent promoter was tolerated, whereas expression from both stronger promoters was lethal. Thus, specific E2F-dependent promoters allow adjustment of p53 expression to selectively induce apoptosis in TCC vs. normal uroepithelial cells. This approach could be useful in targeting apoptosis to TCC and other carcinomas lacking p53 and RB function.     

p73 loss triggers conversion to squamous cell carcinoma reversible upon reconstitution with TAp73alpha

The expression level of the p53 family member, p73, is frequently deregulated in human epithelial cancers, correlating with tumor invasiveness, therapeutic resistance, and poor patient prognosis. However, the question remains whether p73 contributes directly to the process of malignant conversion or whether aberrant p73 expression represents a later selective event to maintain tumor viability. We explored the role of p73 in malignant conversion in a clonal model of epidermal carcinogenesis. Whether sporadic or small interfering RNA (siRNA) induced, loss of p73 in initiated p53+/+ keratinocytes leads to loss of cellular responsiveness to DNA damage by ionizing radiation (IR) and conversion to squamous cell carcinoma (SCC). Reconstitution of TAp73alpha but not DeltaNp73alpha reduced tumorigenicity in vivo, but did not restore cellular sensitivity to IR, uncoupling p73-mediated DNA damage response from its tumor-suppressive role. These studies provide direct evidence that loss of p73 can contribute to malignant conversion and support a role for TAp73alpha in tumor suppression of SCC. The results support the activation of TAp73alpha as a rational mechanism for cancer therapy in solid tumors of the epithelium.