ZNF313 is a novel cell cycle activator with an E3 ligase activity inhibiting cellular senescence by destabilizing p21(WAF1.).

ZNF313 encoding a zinc-binding protein is located at chromosome 20q13.13, which exhibits a frequent genomic amplification in multiple human cancers. However, the biological function of ZNF313 remains largely undefined. Here we report that ZNF313 is an ubiquitin E3 ligase that has a critical role in the regulation of cell cycle progression, differentiation and senescence. In this study, ZNF313 is initially identified as a XIAP-associated factor 1 (XAF1)-interacting protein, which upregulates the stability and proapoptotic effect of XAF1. Intriguingly, we found that ZNF313 activates cell cycle progression and suppresses cellular senescence through the RING domain-mediated degradation of p21(WAF1). ZNF313 ubiquitinates p21(WAF1) and also destabilizes p27(KIP1) and p57(KIP2), three members of the CDK-interacting protein (CIP)/kinase inhibitor protein (KIP) family of cyclin-dependent kinase inhibitors, whereas it does not affect the stability of the inhibitor of CDK (INK4) family members, such as p16(INK4A) and p15(INK4B). ZNF313 expression is tightly controlled during the cell cycle and its elevation at the late G1 phase is crucial for the G1-to-S phase transition. ZNF313 is induced by mitogenic growth factors and its blockade profoundly delays cell cycle progression and accelerates p21(WAF1)-mediated senescence. Both replicative and stress-induced senescence are accompanied with ZNF313 reduction. ZNF313 is downregulated during cellular differentiation process in vitro and in vivo, while it is commonly upregulated in many types of cancer cells. ZNF313 shows both the nuclear and cytoplasmic localization in epithelial cells of normal tissues, but exhibits an intense cytoplasmic distribution in carcinoma cells of tumor tissues. Collectively, ZNF313 is a novel E3 ligase for p21(WAF1), whose alteration might be implicated in the pathogenesis of several human diseases, including cancers.

Opposite functions of HIF-α isoforms in VEGF induction by TGF-ß1 under non-hypoxic conditions.

Transforming growth factor (TGF)-ß1 has biphasic functions in prostate tumorigenesis, having a growth-inhibitory effect in the early stages, but in the late stages promoting tumor angiogenesis and metastasis. We demonstrate here that tumor-producing TGF-ß1 induces vascular endothelial growth factor (VEGF) in prostate cancer cells, and hypoxia-inducible factor (HIF)-1α and HIF-2α has opposite functions in TGF-ß1 regulation of VEGF expression under non-hypoxic conditions. The promoter response of VEGF to TGF-ß1 was upregulated by the transfection of HIF-2α or siHIF-1α but downregulated by HIF-1α and siHIF-2α. Both HIF-1α and HIF-2α were induced by TGF-ß1 at mRNA and protein levels, however, their nuclear translocation was differentially regulated by TGF-ß1, suggesting its association with their opposite effects. VEGF induction by TGF-ß1 occurred in a Smad3-dependent manner, and the Smad-binding element 2 (SBE2, -992 to -986) and hypoxia response element (-975 to -968) in the VEGF promoter were required for the promoter response to TGF-ß1. Smad3 cooperated with HIF-2α in TGF-ß1 activation of VEGF transcription and Smad3 binding to the SBE2 site was greatly impaired by knockdown of HIF-2α expression. Moreover, the VEGF promoter response to TGF-ß1 was synergistically elevated by co-transfection of Smad3 and HIF-2α but attenuated by HIF-1α in a dose-dependent manner. Additionally, TGF-ß1 was found to increase the stability of VEGF transcript by facilitating the cytoplasmic translocation of a RNA-stabilizing factor HuR. Collectively, our data show that tumor-producing TGF-ß1 induces VEGF at the both transcription and post-transcriptional levels through multiple routes including Smad3, HIF-2α and HuR. This study thus suggests that autocrine TGF-ß1 production may contribute to tumor angiogenesis via HIF-2α signaling under non-hypoxic conditions, providing a selective growth advantage for prostate tumor cells.

TRIM72 negatively regulates myogenesis via targeting insulin receptor substrate-1.

Lipid rafts have been known to be platforms to initiate cellular signal transduction of insulin-like growth factor (IGF) inducing skeletal muscle differentiation and hypertrophy. Here, tripartite motif 72 (TRIM72), with a really interesting new gene (RING)-finger domain, a B-box, two coiled-coil domains, and a SPRY (SPla and RYanodine receptor) domain, was revealed to be predominantly expressed in the sarcolemma lipid rafts of skeletal and cardiac muscles. Adenoviral TRIM72 overexpression prevented but RNAi-mediated TRIM72 silencing enhanced C2C12 myogenesis by modulating the IGF-induced insulin receptor substrate-1 (IRS-1) activation through the molecular association of TRIM72 with IRS-1. Furthermore, myogenic activity was highly enhanced with increased IGF-induced Akt activation in the satellite cells of TRIM72(-/-) mice, compared to those of TRIM72+/+ mice. Because TRIM72 promoter analysis shows that two proximal E-boxes in TRIM72 promoter were essential for MyoD- and Akt-dependent TRIM72 transcription, we can conclude that TRIM72 is a novel antagonist of IRS-1, and is essential as a negative regulator of IGF-induced muscle differentiation.

Promoter CpG hypermethylation and downregulation of XAF1 expression in human urogenital malignancies: implication for attenuated p53 response to apoptotic stresses.

XIAP-associated factor 1 (XAF1) is a new candidate tumor suppressor, which has been known to exert proapoptotic effects by interfering with the caspase-inhibiting activity of XIAP. To explore the XAF1's candidacy for a suppressor in urogenital tumorigenesis, we investigated the XAF1 status in a series of cancer cell lines and primary tumors derived from the bladder, kidney and prostate. Expression of XAF1 transcript was undetectable or extremely low in 60% (3/5) of bladder, 66% (10/15) of kidney, and 100% (3/3) prostate cancer cell lines. Abnormal reduction of XAF1 was also found in 33% (18/55) of primary bladder and 40% (8/20) of primary kidney tumors, and showed a correlation with advanced stage and high grade of bladder tumor. Hypermethylation at 14 CpG sites in the 5' proximal region of the XAF1 promoter was highly prevalent in cancers versus adjacent normal or benign tissues and tightly associated with reduced gene expression. XAF1 expression enhanced the apoptotic response of tumor cells to chemotherapeutic agents, such as etoposide or 5-FU. While XAF1 expression did not influence the subcellular distribution or expression of XIAP, it elevated the protein stability of p53 and its target gene expression. Moreover, the apoptosis-sensitizing and growth suppression function of XAF1 was markedly impeded by blockade of p53 function. Collectively, our study demonstrates that epigenetic alteration of XAF1 is frequent in human urogenital cancers and may contribute to the malignant progression of tumors by rendering tumor cells a survival advantage partially through the attenuated p53 response to apoptotic stresses.

Expression and mutation analyses of MKK4, a candidate tumour suppressor gene encoded by chromosome 17p, in human gastric adenocarcinoma.

Homozygous deletion or somatic mutations of mitogen-activated protein kinase kinase 4 (MKK4), a candidate tumour suppressor gene located at 17p11, have been observed in many types of human tumours. To explore the likelihood that MKK4 acts as a suppressor in gastric tumorigenesis, we examined the expression and mutation status of MKK4 in 144 gastric tissues and cell line specimens. Expression of the MKK4 transcript was easily detectable in all normal and benign tumour tissues and none of 102 primary carcinomas and cell lines showed an abnormal reduction in MKK4 expression. Expression levels of MKK4 transcript showed no cancer-specific reduction in 43 matched sets and did not correlate with stage, grade and histopathological types of the tumours. Western blot analysis also revealed that MKK4 protein expression in carcinoma tissues and cell lines is comparable to non-cancerous tissues. A significant loss of heterozygosity (LOH) was detected at telomeric markers of the MKK4, locus. However, no allelic deletion of the MKK4 gene or at the centromeric loci was identified. Moreover, no evidences for somatic mutations leading to amino acid substitutions or frameshifts of MKK4 were identified in the carcinoma tissues and cell lines, whereas a substantial fraction of the same set showed allelic loss or mutations of the TP53 gene located at 17p13, suggesting that LOH at telomeric loci or the TP53 locus might not extend into the MKK4 gene in gastric cancers. In this study, we also report the identification of a highly conserved MKK4 processed pseudogene, which shares 95% homology with the coding region of the functional MKK4 transcript. Collectively, our data demonstrate that genomic deletion or somatic mutation of MKK4 is infrequent in gastric cancers, suggesting that MKK4 might not be a critical target of genetic inactivation in gastric tumorigenesis.

Frequent epigenetic inactivation of RASSF1A by aberrant promoter hypermethylation in human gastric adenocarcinoma.

Methylation associated inactivation of RASSF1, a putative tumor suppressor identified at 3p21.3, has been frequently observed in several human malignancies, including lung and breast cancers. To explore the penetrance of RASSF1 in gastric carcinogenesis, we performed expression and mutation analyses of 3 isotypes of RASSF1 (A, B, and C) in 150 gastric specimens, including 15 carcinoma cell lines. RASSF1A and RASSF1B transcripts were not expressed in 60% (9 of 15) and 33% (5 of 15) of gastric carcinoma cell lines, respectively, whereas RASSF1C was detectable in all cell lines. Bisulfite DNA sequencing analysis revealed that the CpG island in the RASSF1A promoter is hypermethylated in all RASSF1A-nonexpressing cell lines. In addition, both RASSF1A and RASSF1B were re-expressed by treatment with the demethylating agent 5-aza-2'-deoxycytidine. Among 90 primary gastric adenocarcinomas examined, 41 (46%) and 19 (21%) expressed no or abnormally low levels of RASSF1A and RASSF1B, respectively, and 12 (13%) tumors showed no expression of both isoforms. Loss or abnormal down-regulation of RASSF1A correlated with tumor stage and grade but not with histological types of tumors. Methylation-specific PCR analysis demonstrated that 95% (39 of 41) of RASSF1A-nonexpressing primary tumors are methylated at the CpG sites in the promoter, whereas none of the adjacent noncancerous or normal tissues are methylated. No somatic mutations were detected in RASSF1 transcripts expressed in unmethylated tumors. However, 10 methylated tumors, including 4 cell lines, showed low genomic levels of RASSF1 and expressed no RASSF1A transcripts, suggesting that RASSF1A inactivation might be caused by both epigenetic and genetic mechanisms in a subset of gastric adenocarcinomas. In conclusion, our data indicate that epigenetic transcriptional silencing of RASSF1, especially RASSF1A isoform, is a frequent event in gastric tumorigenesis and might play an important role in the malignant progression of gastric adenocarcinomas.

Absence of Bcl10 gene mutations in Ewing's sarcomas.

The Bcl10 gene was recently isolated from the breakpoint region of t(1;14)(p22;q32) in mucosa-associated lymphoid tissue (MALT) lymphomas. Somatic mutations of Bcl10 were found in not only t(1;14)-bearing MALT lymphomas, but also a wide range of other tumors. To clarify the actual frequency and spectrum of Bcl10 mutations in primary malignant Ewing's sarcomas, we examined 31 cases of Ewing's sarcomas. Polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP) and sequencing analyses were done. No Bcl10 mutations were found in our series of Ewing's sarcomas. While screening for mutations, we found three polymorphisms at codons 8 exon 1 of the Bcl10 gene. Our results strongly suggest that somatic mutations of Bcl10 are extremely rare in Ewing's sarcomas and do not commonly contribute to their molecular pathogenesis.

Frequent epigenetic inactivation of RASSF1A in human bladder carcinoma.

Allelic deletion or transcriptional silencing of RASSF1, a putative tumor suppressor at 3p21.3, has been found in a considerable proportion of lung, breast, and ovarian cancers. In this study, we analyzed the expression and mutation status of three RASSF1 isoforms (-A, -B, and -C) in 55 primary bladder carcinomas and 10 bladder and prostate cancer cell lines. The RASSF1A transcript was not found in 80% (4 of 5) and 100% (4 of 4) of bladder and prostate cell lines, respectively. Compared with normal bladder tissues, loss or significant reduction of RASSF1A was identified in 62% (34 of 55) of primary bladder carcinomas and 10 (83%) of 12 matched sets showed tumor-specific alteration of RASSF1A expression. Moreover, loss or abnormal down-regulation of RASSF1A correlated with advanced tumor stage. RASSF1B was undetectable in 60% (3 of 5) of bladder cell lines and in 31% (17 of 55) of primary tumors, but none of these tumors showed altered expression exclusively in RASSF1B. RASSF1C transcript was detected in all cell lines and primary tumors we examined. Expression of RASSF1A and RASSF1B was reactivated in all nonexpressor cell lines by treatment with the demethylating agent 5-aza-2'-deoxycytidine. Bisulfite DNA sequencing analysis revealed that aberrant hypermethylation at the CpG island in the RASSF1A promoter is strongly associated with the loss of RASSF1A expression in cell lines and uncultured primary tumors. Methylation-specific PCR and BstUI digestion analyses also demonstrated that 97% (33 of 34) of RASSF1A-nonexpressing primary tumors are methylated. Although somatic mutations were not identified in RASSF1 transcripts expressed in unmethylated tumors, 24% (9 of 37) of methylated cell lines and primary tumors showed detectable reductions in genomic levels of RASSF1, suggesting that RASSF1A inactivation might be caused by both epigenetic and genetic mechanisms in a subset of bladder tumors. Together, our data suggest that RASSF1A inactivation may play a critical role in the malignant progression of human bladder carcinomas.

Low incidence of genetic alterations of the p16CDKN2a in clear cell chondrosarcoma.

Mutational inactivation of the cyclin-dependent kinase inhibitors (CDKIs) (p16CDKN2a) tumor suppressor gene has been found in a variety of human tumor types. To investigate the involvement of CDKI abnormality in clear cell chondrosarcoma, alterations of CDKIs were examined in clear cell chondrosarcoma tissues using a quantitative DNA/PCR, PCR-SSCP. Two of 38 specimens (5.2%) we analyzed showed abnormally low levels of p16CDKN2a amplification, suggesting that the allelic deletion of the gene might be low frequent event in progression of this tumor. For detection of subtle sequence alterations such as point mutations, we performed SSCP analysis of the entire coding region of the p16CDKN2a gene. No altered SSCP patterns were found in 38 clear cell chondrosarcoma specimens. This study reflects the very low incidence of genetic alterations of the p16CDKN2a gene in clear cell chondrosarcoma. Therefore, we conclude that the alteration of the p16CDKN2a gene is not involved significantly in the development of clear cell chondrosarcoma.

Overexpression of p53 and absent genetic mutation in clear cell chondrosarcoma.

Clear cell chondrosarcoma is one of the extremely rare chondrosarcomas. The pathogenesis and the molecular genetic events, which contribute to the development of clear cell chondrosarcoma, are not well elucidated, due in part to the lack of sufficient tumor tissue available. To characterize the involvement of the p53 gene abnormality in this disease, we analyzed expression and sequence alteration of p53 by immunohistochemical analysis of the protein expression and quantitative DNA/PCR and PCR-SSCP assays of the gene in 28 paraffin-embedded tissue specimens. Immunohistochemical analysis demonstrated that 7 (25%) showed patchy positive nuclear staining for p53 and 5 (18%) showed diffuse positive nuclear staining patterns. Sixteen (57%) were negative for p53 immunostaining. Quantitative DNA/PCR analysis revealed that none of the cases we studied showed significantly reduced levels of p53 amplification (<0.50), strongly suggesting an allelic deletion of the p53 gene. In contrast, however, DNA/PCR-SSCP analysis failed to detect any types of mutations resulting in amino acid substitution within exons 5-9 regions of the gene. Taken together, our data suggest that genetic alteration of p53 is a relatively rare event in clear cell chondrosarcomas but a substantial fraction of this type of tumors carries abnormal overexpression of p53, which might result from an as yet unidentified mechanism(s).

Increased expression of cFLIP(L) in colonic adenocarcinoma.

During tumour progression, cancer cells use diverse mechanisms to escape from apoptosis-inducing stimuli, which may include receptor internalization, inhibition of signal pathways, and regulation of specific sets of genes. Substantial numbers of colon cancer cells have been observed to express Fas/Fas ligand, but are resistant to Fas-mediated apoptosis, suggesting that colonic tumours might develop specific mechanisms to overcome Fas-mediated apoptosis. Recently, cellular FLICE-like inhibitory protein (cFLIP) has been identified as an endogenous inhibitor of Fas- or other receptor-mediated apoptosis and its altered high expression has a suspected association with tumour development or progression. In an effort to investigate the prevalence of cFLIP(L) alterations in colon carcinomas and their possible implications for the progression of colon cancers, cFLIP(L) expression was analysed in adenocarcinomas and adenomatous polyps of colon, with matched normal tissues, at RNA and protein levels, by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. cFLIP(L) transcripts were constitutively expressed in colon cancers and expression levels were significantly higher in carcinomas than in normal tissues (p<0.05). Overexpression of cFLIP(L) protein was found exclusively in carcinoma cells in all matched sets analysed and approximately three-fold induction was detected in cancer cells (p<0.05). The expression of cFLIP(L) protein was not significantly altered in adenomatous polyps compared with normal tissues. Taken together, these results strongly suggest that abnormal overexpression of cFLIP(L) is a frequent event in colon carcinomas and might contribute to in vivo tumour transformation.

P53 mutations in Ewing's sarcoma.

The p53 tumor suppressor gene is one of the most frequently altered genes in human malignancies. To explore the implication of p53 alteration in Ewing's sarcoma, we analyzed the deletion and sequence alterations of p53 and abnormal amplification of MDM2, which acts as a functional inhibitor of p53, in 35 tissue specimens. Quantitative genomic PCR analysis showed that 2 of 35 tumors have extremely low levels of the p53 gene, indicating a homozygous deletion of the gene. Mutational analysis of exons 4 to 9 of p53 by PCR-SSCP revealed that 3 of 35 tumors carry sequence alterations in exons 5 or 8, and DNA sequencing analysis identified missense point mutations at codon 132 (AAG-->ATG, lysine-->methionine) and codon 135 (TGC-->TCC, cystein-->serine) in exon 5, and codon 287 (GAG-->GTG, glutamic acid-->valine) in exon 8 from these tumors. No abnormal amplification of the MDM2 gene was recognized. Taken together, our data demonstrate that p53 is genetically altered in a small fraction of Ewing's sarcoma.

Costunolide induces apoptosis by ROS-mediated mitochondrial permeability transition and cytochrome C release.

Costunolide is an active compound isolated from the root of Saussurea lappa Clarks, a Chinese medicinal herb, and is considered a therapeutic candidate for various types of cancers. Nevertheless, the pharmacological pathways of costunolide are still unknown. In this study, we investigate the effects of costunolide on the induction of apoptosis in HL-60 human leukemia cells and its putative pathways of action. Using apoptosis analysis, measurement of reactive oxygen species (ROS), and assessment of mitochondrial membrane potentials, we show that costunolide is a potent inducer of apoptosis, and facilitates its activity via ROS generation, thereby inducing mitochondrial permeability transition (MPT) and cytochrome c release to the cytosol. ROS production, mitochondrial alteration, and subsequent apoptotic cell death in costunolide-treated cells were blocked by the antioxidant N-acetylcystein (NAC). Cyclosporin A, a permeability transition inhibitor, also inhibited mitochondrial permeability transition and apoptosis. Our data indicate that costunolide induces the ROS-mediated mitochondrial permeability transition and resultant cytochrome c release. This is the first report on the mechanism of the anticancer effect of costunolide.

Mutational alterations of the p16CDKN2A tumor suppressor gene have low incidence in mesenchymal chondrosarcoma.

Mutational inactivation of the cyclin-dependent kinase inhibitors (CDKIs) (p16INK4A/MTS1) tumor suppressor gene has been found in a variety of human tumor types. To investigate the involvement of CDKI abnormality in mesenchymal chondrosarcoma, alterations of CDKIs were examined in human mesenchymal chondrosarcoma tissues using a quantitative DNA/PCR, PCR-SSCP. Seven of 33 specimens (21.2%) showed abnormally low levels of p16CDKN2A amplification, suggesting that the allelic deletion of the gene might be a less frequent event in progression of this tumor. To detect subtle sequence alterations such as point mutations, SSCP analysis of the entire coding region of the p16CDKN2A gene, exons 1, 2, and 3 regions, showed no altered SSCP patterns in 33 mesenchymal chondrosarcoma specimens. A low incidence of genetic alterations of the p16CDKN2A was found in mesenchymal chondrosarcoma. Through this study, we conclude that alteration of the p16CDKN2A gene does not participate significantly in the tumorigenesis of mesenchymal chondrosarcoma.

Overexpression of p53 and rare genetic mutation in mesenchymal chondrosarcoma.

Mesenchymal chondrosarcoma is extremely rare and accounts for less than 2% of all chondrosarcomas. The pathogenesis and the molecular genetic events which contribute to the development of mesenchymal chondrosarcoma are not well elucidated, due in part to the lack of sufficient tumor tissue available. To characterize the involvement of the p53 gene abnormality in this disease, we analyzed expression and sequence alteration of p53 by immunohistochemical analysis of the protein expression and quantitative DNA/PCR and PCR-SSCP assays of the gene in 33 paraffin-embedded tissue specimens. Immunohistochemical analysis demonstrated that 19 (61.3%) of 31 had nuclear overexpression of p53 while 7 (22.6%) showed cytoplasmic expression. The remaining 5 (16.1%) were negative for p53 staining. The nuclear positivity of p53 was observed within a range of 22-64% (mean 37.3%) of tumor cells and showed a positive staining in mesenchymal components as well as chondroid components. Quantitative DNA/PCR analysis revealed that 6 (18.2%) of the 33 specimens carried significantly reduced or undetectably low levels of p53 indicating the genomic deletion of the gene in these tumors. In contrast, however, DNA/PCR-SSCP analysis failed to detect any types of mutations resulting in amino acid substitution within exons 5-9 regions of the gene. Taken together, our data suggests that genetic alteration of p53 is a relatively rare event in mesenchymal chondrosarcomas but substantial fraction of this type of tumors carries abnormal overexpression of p53, which might result from as yet unidentified epigenetic mechanism(s).

Frequent alteration of p63 expression in human primary bladder carcinomas.

p63, a recently identified member of the p53 gene family, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. To explore the penetrance of p63 in bladder carcinogenesis, we performed expression and mutation analyses of two major isotypes, TAp63 and deltaNp63, in 63 bladder specimens. In 12 normal tissues, TAp63 was expressed at an easily detectable level whereas deltaNp63 was absent or extremely low. While none of 47 carcinomas showed allelic deletion of the gene, marked reduction of TAp63 and abnormal overexpression of deltaNp63 were found in 25 (53.2%) and 30 (63.8%) carcinomas, respectively. Tumor-specific alteration of TAp63 and deltaNp63 expression was identified in two and three of six matched sets, respectively. In addition, reduced expression of TAp63 showed a correlation with tumor stage and grade. Abnormal expression of TAp63 or deltaNp63 isoform was also observed in three of four cell lines, and treatment with 5-Aza-2'-deoxycytidine led to up- or down-regulation of TAp63 and/or deltaNp63 expression, suggesting that the promoters of both isoforms might be affected by DNA methylation, but not in a reciprocal fashion. No sequence alteration of p63 was identified in 47 carcinomas whereas 17 (34.8%) of these showed p53 mutations, and no association between p63 expression and the mutational status of p53 or expression of p21Waf1, MDM2, and 14-3-3sigma was recognized. Our data suggest that altered expression of p63 is a frequent event in bladder carcinogenesis and might contribute to the progression of bladder tumors, possibly via the mechanism(s) distinct from the p53 pathway.

Mitogenic conversion of transforming growth factor-beta1 effect by oncogenic Ha-Ras-induced activation of the mitogen-activated protein kinase signaling pathway in human prostate cancer.

Elevated expression of transforming growth factor (TGF)-beta1 has been implicated in prostate tumorigenesis despite its growth-inhibitory effect on normal epithelial and carcinoma cells of the prostate. In this study, we identified that G1-to-S transition of the cell cycle is stimulated by TGF-beta1 in the prostate cancer cell line TSU-Pr1. No mutation of signal mediators, including Smads, and induction of PAI-1 transcription indicated that the TGF-beta1 signaling cascade is functionally intact in this cell line. Whereas pharmacological inhibitors of various mitogenic signaling pathways showed no effects, blockade of the mitogen-activated protein kinase (MAPK) pathway by the MAPK kinase 1 inhibitor PD98059 restored the growth inhibitory role of TGF-beta1 in TSU-Pr1, which carries an oncogenic mutation in Ha-Ras (V12). Moreover, expression of antisense Ha-Ras or dominant negative Raf-1 abrogated the mitogenic effect of TGF-beta1 in TSU-Pr1, and the TGF-beta1 inhibition of DU145 was switched to stimulation by V12Ha-Ras transfection. Whereas the negative growth regulation by TGF-beta1 was completely inhibited by dominant negative Smad2, Smad3, or Smad4, its mitogenic effect was not affected, suggesting that this action is Smad-independent. Interestingly, whereas the TGF-beta1-mediated up-regulation of p15INK4B and p21WAF1 transcription was abolished in TSU-Pr1 and V12Ha-Ras-transfected DU145, inhibition of the Ras/MAPK pathway restored the TGF-beta1 induction of these genes. Taken together, our data suggest that prostate carcinomas with the Ras/MAPK pathway activation might have a selective growth advantage by autocrine TGF-beta1 production.

Loss of imprinting and elevated expression of wild-type p73 in human gastric adenocarcinoma.

The p73 gene located at 1p36.3 encodes for a protein with significant similarity to p53. To investigate the penetrance of p73 in gastric carcinogenesis, we analyzed the expression, allelotype, and mutation of p73 in five cell lines and 75 tissues. Although extremely low levels of p73 expression were observed in all noncancerous gastric tissues and four of five cell lines, a significant elevation of p73 was detected in 37 of 39 (94.9%) carcinoma tissues. Furthermore, a tumor-specific increase of p73 was identified in 14 of 16 (87.5%) matched sets. Allelotyping analysis using a StyI or BanI polymorphism revealed that 5 of 21 (23.8%) informative carcinomas, but none of 19 noncancerous cases, express p73 biallelically, suggesting the transcriptional activation of a silent allele in a subset of cancers. Whereas the transcription of an active allele was markedly induced by serum starvation or clump formation of the cells, treatment with 5-aza-2'deoxycytidine activated a silent allele with a subsequent up-regulation of an active allele, supporting the genomic imprinting and autoregulation of the gene. Allelic deletion or mutation of the gene was not found, and no association of p73 expression with the mutational status of p53 or expression of p21Waf1 was recognized. Taken together, this study argues that p73 is not a target of genetic alteration in gastric carcinogenesis and suggests that overexpression of p73 might be triggered by physiological stresses accompanied with outgrowth of tumors, such as hypoxia or nutrient deprivation.

TGF-beta1 inhibition of apoptosis through the transcriptional up-regulation of Bcl-X(L) in human monocytic leukemia U937 cells.

To characterize the TGF-beta1 response of monocytic leukemia cells, we analyzed the effects of TGF-beta1 on cell proliferation, differentiation, and apoptosis of human monoblastic U937 cells. Treatment of cells with TGF-beta1 in the absence of growth factors significantly enhanced cell viability. Flow cytometric analysis of DNA content and CD14 expression revealed that TGF-beta1 does not affect cell proliferation and differentiation. Consistent with these results was the finding that no transcriptional induction of Cdk inhibitors such as p21Waf1, p15Ink4b, and p27Kip1 was detected following TGF-beta1 treatment. Interestingly, however, pretreatment of TGF-beta1 significantly inhibited Fas-, DNA damage-, and growth factor deprivation-induced apoptosis. This antiapoptotic effect was totally abrogated by anti-TGF-beta1 antibody. Quantitative RT-PCR analysis demonstrated a dose- and time-dependent transcriptional up-regulation of Bcl-X(L), suggesting its implication in the TGF-1-mediated antiapoptotic pathway. We also observed elevated expression of c-Fos and PTEN/MMAC1. But, no detectable change was recognized in expression of c-Jun, Fas, Fadd, Fap-1, Bcl-2, and Bax. Taken together, our study shows that TGF-beta1 enhancement of cellular viability is associated with its antiapoptotic effect, which may result from the transcriptional up-regulation of Bcl-X(L).

Mutational alteration of the p16CDKN2a tumor suppressor gene is infrequent in Ewing's sarcoma.

Mutational alteration of the p16CDKN2a tumor suppressor gene has been frequently observed in a variety of human cell lines and tumor tissues. To assess whether alterations of the p16CDKN2a gene play an important role in the pathogenesis of Ewing's sarcoma, we examined the allelic deletion and point mutation of the gene in 27 primary tumors. In quantitative DNA/PCR analysis for three individual exons of the gene, none of the 27 specimens showed detectable reduction in the amplification levels compared to those of normal lymphocytes. To explore the presence of any subtle sequence changes within the protein coding region, we performed a comprehensive screening of sequence alteration in the three exons using DNA/PCR-SSCP analysis. However, no abnormal shift of SSCP bands indicative of sequence change was identified. In addition, no elevation of p16CDKN2a mRNA expression was observed in the RD-ES cell line following 5'-Aza-cytidine treatment, indicating that the promoter of the gene is not abnormally methylated in this cell line. Immunohistochemical study of the same tissue specimens for p16CDKN2a and pRB also revealed grade 2+ or 3+ nuclear staining of both proteins in most of the specimens we examined. Taken together, our results strongly suggest that the mutational inactivation of the p16CDKN2a gene might be a rare event, and thus not play a critical role in the pathogenesis of Ewing's sarcoma.