CCN3 is a novel endogenous PDGF-regulated inhibitor of glomerular cell proliferation.

CCN proteins affect cell proliferation, migration, attachment, and differentiation. We identified CCN3 as a suppressed gene following platelet-derived growth factor (PDGF)-BB or -DD stimulation in a cDNA-array analysis of mesangial cells. In vitro growth-arrested mesangial cells overexpressed and secreted CCN3, whereas the addition of the recombinant protein inhibited cell growth. Induction of mesangial cell proliferation by PDGF-BB or the specific PDGF beta-receptor ligand PDGF-DD led to downregulation of CCN3 mRNA, confirming the array study. Specific PDGF alpha-receptor ligands had no effect. CCN3 protein was found in arterial smooth muscle cells, the medullary interstitium, and occasional podocytes in the healthy rat kidney. Glomerular CCN3 was low prior to mesangial proliferation but increased as glomerular cell proliferation subsided during mesangioproliferative glomerulonephritis (GN). Inhibition of PDGF-B in mesangioproliferative disease led to overexpression of glomerular CCN3 mRNA. CCN3 localized mostly to podocytes in human glomeruli, but this expression varied widely in different human glomerulonephritides. Glomerular cell proliferation negatively correlated with CCN3 expression in necrotizing GN. Our study identifies CCN3 as an endogenous inhibitor of mesangial cell growth and a modulator of PDGF-induced mitogenesis.

Y-box protein-1 controls transforming growth factor-beta1 translation in proximal tubular cells.

Transforming growth factor-beta1 (TGF-beta1) mRNA has low basal translational efficiency in proximal tubule cells; however, its translation is stimulated by profibrotic cytokines. We studied the role of the multifunctional Y-box protein-1 (YB-1) in regulating proximal tubule cell TGF-beta1 translation. Using RNA-electrophoretic mobility shift assays and ultraviolet crosslinking, we found two protein complexes of 50 and 100 kDa, which bound to the TGF-beta1 mRNA 5'-untranslated region. Supershift studies using antibodies to YB-1 showed that both sites contained YB-1 as did studies with recombinant YB-1, which demonstrated that it was sufficient to form both complexes. RNA competition experiments confirmed YB-1 binding to the two predicted binding sites; one with high affinity and the other with lower affinity. Strong basal YB-1 association with TGF-beta1 mRNA was found in proximal tubule cells, which decreased when platelet-derived growth factor was used to activate TGF-beta1 translation. In contrast, knockdown of proximal tubule cell YB-1 expression abrogated TGF-beta1 synthesis. Our results suggest that TGF-beta1 translation in proximal tubule cells requires YB-1 binding to a high-affinity site in the 5'-untranslated region of its mRNA; however, binding to a low-affinity site inhibits basal translation.

Biological responses to PDGF-BB versus PDGF-DD in human mesangial cells.

Platelet-derived growth factor (PDGF)-BB and PDGF-DD mediate mesangial cell proliferation in vitro and in vivo. While PDGF-BB is a ligand for the PDGF alpha- and beta-receptor chains, PDGF-DD binds more selectively to the beta-chain, suggesting potential differences in the biological activities. Signal transduction and regulation of gene expression induced by PDGF-BB and -DD were compared in primary human mesangial cells (HMCs), which expressed PDGF alpha- and beta-receptor subunits. The growth factor concentrations used were chosen based on their equipotency in inducing HMCs proliferation and binding to the betabeta-receptor. Both growth factors, albeit at different concentrations induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. In addition, PDGFs led to the phosphorylation and activation of signal transducers and activators of transcription 1 (STAT1) and STAT3. HMCs proliferation induced by either PDGF-BB or -DD could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase-, Janus kinase (JAK)/STAT-, or phosphatidyl-inositol 3-kinase pathways. Using a gene chip array and subsequent verification by real-time reverse transcriptase (RT)-polymerase chain reaction, we found that in HMC genes for matrix metalloproteinase 13 (MMP-13) and MMP-14 and, to a low extent, cytochrome B5 and cathepsin L were exclusively regulated by PDGF-BB, whereas no exclusive gene regulation was detected by PDGF-DD. However, at the protein level, both MMP-13 and -14 were equally induced by PDGF-BB and -DD. PDGF-BB and -DD effect similar biological responses in HMCs albeit at different potencies. Rare apparently differential gene regulation did not result in different protein expression, suggesting that in HMCs both PDGFs exert their biological activity almost exclusively via the PDGF beta-receptor.

Predictive value of molecular alterations for the prognosis of urothelial carcinoma.

Accumulation of p53 and C-Myc overexpression are frequently found in advanced urothelial carcinomas. The prevalence and predictive value of both molecular alterations was investigated in 61 patients with superficial urothelial tumors. Distinct patterns of p53 accumulation and C-Myc overexpression were observed in superficial urothelial carcinoma of different stages. For instance, 67% of carcinomata in situ displayed accumulation of p53, but only 44% showed C-Myc overexpression, whereas in pT1 tumors the corresponding percentages were 25 and 75%. Similarly, while p53 accumulation was significantly (p = 0.02) associated with tumor grade, C-Myc overexpression did not correlate with grade. In multivariate analysis, p53 accumulation was found to be an independent predictor of tumor progression (p = 0.0096), whereas C-Myc overexpression did not correlate with the course of disease. Alterations in both markers together predicted neither tumor recurrence nor tumor progression better than p53 accumulation on its own. Sufficient expression of C-Myc may be a general requirement for proliferative competence in urothelial tumors, barring its use as a predictive marker. The predictive value of p53 accumulation for tumor progression was further underlined by the finding that in a distinct group of 52 patients with progressive urothelial carcinoma 73% of the recurrent tumors displayed p53 accumulation.

Patterns of allelic loss on chromosome 17 in sporadic breast carcinomas detected by fluorescent-labeled microsatellite analysis.

Loss of heterozygosity (LOH) on chromosome 17 is a frequent genetic alteration in breast cancer. To assess whether the location of potential tumor suppressor genes is compatible with the LOH pattern in individual tumors, we analyzed allele loss on chromosome 17 in 121 invasive ductal breast carcinomas and 16 benign breast tumors with 14 polymorphic microsatellite markers (4 on 17p and 10 on 17q). Fluorescent polymerase chain reaction (PCR) for typing microsatellites coupled with DNA fragment analysis in an automated DNA sequencer was applied. Frequencies of LOH varied from 29.4% (D17S1322) to 57.4% (TP53-Alu). No LOH could be detected in benign breast tumors. In 54 tumors the deletion patterns were consistent with the complete loss of 17p (n = 28), 17q (n = 9) or the whole chromosome 17 (n = 17). Five smallest regions of overlap (SROs) were identified in tumors with interstial deletion patterns. On 17p, two foci were detected affecting the TP53 locus and the hypermethylated in cancer I (HICI) region (17p13.3). On 17q, SRO1 was localized between markers THRAI and D17S855, centromeric to the breast/ovarian cancer gene BRCAI; SRO2 was flanked by markers AFM234 and NMEI, and SRO3 was centered between markers MPO and GH. Associations between LOH and histopathological characteristics were determined. Significant correlations were found between higher grade and loss of the TP53 gene (marker TP53, P = 0.019), loss of the BRCAI region (P < 0.009), LOH of marker AFM155 (P = 0.003) and marker NMEI (P = 0.026). For positive estrogen receptor status, only LOH of the THRAI marker correlated significantly, whereas highly significant correlations were determined between positive progesterone receptor and markers centromeric to the BRCAI region D17S250 (P = 0.00002), THRAI (P = 0.0006), and the intragenic BRCAI markers [D17S1322 (P = 0.021), D17S855 (P = 0.029)]. Results presented in this study identify five independent regions of chromosome 17 which are likely to contain potential tumor suppressor genes involved in the carcinogenesis of sporadic breast cancer.

c-myc in bladder cancer. Clinical findings and analysis of mechanism.

The c-myc gene product is known to be involved in the regulation of cell proliferation and differentiation. Altered c-myc gene expression is a common event in a variety of tumors. This study was designed to investigate c-myc overexpression in transitional cell carcinoma (TCC). The first part was designed to investigate the frequency of c-myc overexpression in relation to tumor stage and tumor grade. A second set of experiments was directed at the mechanisms underlying c-myc overexpression in TCC. A total of 185 paraffin-embedded urothelial tissue specimens were investigated immunohistochemically for c-myc overexpression. A single case of overexpression (6%) was observed in normal urothelial tissue (n = 16). c-myc overexpression was also infrequent in carcinoma in situ (TIS) (7/39 = 18%). In contrast, papillary urothelial tumors (n = 65) yielded c-myc overexpression in 38 cases (58%). Investigation of infiltrating bladder tumors revealed c-myc overexpression in 56% of T1 tumors and 59% of muscle-infiltrating tumors. The staining pattern in multifocal tumors was heterogeneous in 10 of 18 cases. Similarly, only 12 of 28 patients with tumor recurrences showed the same c-myc staining pattern in the primary tumor and in tumor recurrences. c-myc overexpression did not correlate with tumor grade or tumor progression. Nevertheless, the high frequency of c-myc overexpression in urothelial carcinoma suggests an important role for this protein in urothelial carcinoma. Therefore, the mechanism underlying c-myc overexpression was further investigated in six bladder carcinoma cell lines. Southern blot experiments under standardized conditions showed no significant gene amplification. The comparison of c-myc mRNA expression to that of histone H3 as a measure of cell proliferation revealed a moderate correlation (r = 0.45) in the six cell lines examined. These data suggest that in accord with its established role as a cell cycle competence factor, c-myc may be necessary but not sufficient for the induction of proliferation in urothelial carcinoma.

Detection of sequential genetic alterations relevant for breast cancer development.

Breast cancer emerges as a multistep process with transformation of normal cells via steps of hyperplasia, premalignant change and in situ carcinoma. Cytogenetic and molecular genetic analyses of breast cancer samples indicate that tumour development involves the accumulation of various genetic alterations, including amplification of oncogenes and mutation or loss of tumour suppressor genes. Microdissection of histological sections is needed to correlate the specific histological change and the genetic alteration. For detection of oncogene amplification quantitative differential polymerase chain reaction (PCR) can be used. For assessment of loss of heterozygosity PCR-based microsatellite polymorphisms detecting differences in short tandem repeat sequences are much more informative than standard two-allele restriction fragment length polymorphism markers. Still, the direct correlation of the genetic alterations to specific histological findings is the key to reveal insight into tumour biology and thereby gain prognostic information for the individual breast cancer patient.

Frequent allele loss on 9p21-22 defines a smallest common region in the vicinity of the CDKN2 gene in sporadic breast cancer.

Genetic studies of chromosome arm 9p have indicated the presence of one or more tumor suppressor genes involved in genetic susceptibility to various types of human cancers. To define the extent of the specific deletion of 9p21-22 in human breast cancer, we have analyzed loss of heterozygosity and homozygous deletion of 9p21-22 in 68 paired blood and tumor samples by using fluorescent multiplex polymerase chain reaction (PCR). Of these tumors, 43% (29/68), including two ductal carcinomas in situ (DCIS), showed allele loss at one or more loci analyzed. Homozygous deletion for 9p markers was detected in 7/68 (10%) of tumor samples. Eleven tumors showed allele loss at all informative loci, and 18 tumors showed selective deletion on 9p21-22. Allele deletions in six tumors did not involve microsatellite markers flanking CDKN2. The smallest common region of deletion could be defined between D9S171 and D9S126. No significant associations were observed between deletion of 9p21-22 and any of the histopathological parameters analyzed. However, the abundance of deletions of this chromosomal region still suggests that loss and inactivation of putative tumor suppressor gene(s) located on 9p21-22 may be involved in the pathogenesis of breast cancer.

Clinical impact of detection of loss of heterozygosity of BRCA1 and BRCA2 markers in sporadic breast cancer.

The development of familial and sporadic breast cancer is based on genetic alterations of tumour-suppressor genes, for which loss of heterozygosity (LOH) is one mechanism of gene inactivation. To investigate LOH of BRCA1 (17q21) and BRCA2 (13-q12-13) in sporadic breast cancer, polymerase chain reaction (PCR)-based fluorescent DNA technology for detection of microsatellite polymorphisms was applied. A total of 137 breast cancer and 15 benign breast specimens with matched normal tissue were examined. Fluorescent-labelled PCR products were analysed in an automated DNA sequencer (ALFTM Pharmacia). Losses at both loci were correlated with different histological types, age, tumour size, lymph node status, grading and steroid hormone receptor expression, [SHR: oestrogen receptor (ER), progesterone receptor (PgR)]. For BRCA1 (D17S855, THRA1, D17S579) losses could be detected in invasive ductal carcinoma (IDC; n = 108) in 32-38%, invasive lobular carcinoma (ILC; n = 19) in 21-42% depending on the marker applied, but not in benign breast tumours (n = 15). Losses of BRCA1 markers correlated with larger tumour size, higher grade, and PgR expression. For BRCA2 (D13S260, D13S267, D13S171) losses could be detected in 108 IDCs in 30-38%, in 19 ILCs in 17-39% depending on the marker applied, but not in benign breast tumours. Losses of BRCA2 markers correlated only with higher grade. Microsatellite analyses combined with detection of fluorescent-labelled PCR products by an automated laser DNA sequencer can be used for routine determination of LOH. In sporadic breast cancer, LOH of BRCA1 of BRCA2 does not add decisive prognostic value as stated for familial breast cancer.

ERBB2 gene amplification detected by fluorescent differential polymerase chain reaction in paraffin-embedded breast carcinoma tissues.

For quantificative determination of ERBB2 gene amplification in archival human carcinoma specimens we have developed a rapid, non-radioactive approach, which is based on the differential polymerase chain reaction (PCR) and fluorescent DNA technique. Sequences from the ERBB2 gene and from a single-copy reference gene were amplified simultaneously by PCR, in which one of each primer pair was fluorescently labelled. PCR products were separated by polyacrylamide gel electrophoresis in an automated DNA sequencer and directly quantified after laser activation and emission scanning using appropriate software. This fluorescent differential polymerase chain reaction (fd-PCR) method was used for quantificative determination of ERBB2 gene amplification in 195 formalin-fixed, paraffin-embedded breast carcinoma tissues. ERBB2 gene amplification was found in 52 (26%) of these tumors and correlated significantly with tumor size, absence of estrogen receptor (ER) and pS2 expression, but not with absence of progesterone receptor (PR) or presence of epidermal growth factor receptor (EGF-R) expression, lymph-node metastases or grading. In univariate analysis, ERBB2 gene amplification showed no significant correlation with clinical outcome, either in the whole population or in the subgroup defined by positive axillary lymph-node metastases. However, within the node-negative subgroup, patients with ERBB2 gene amplification had significantly decreased relapse-free survival and overall survival (p < 0.05). The fd-PCR assay is a valuable tool for determination of amplification of ERBB2 gene as well as further oncogenes. In this way, more detailed information about individual tumor biology may be acquired by a routine assay.

P53 accumulation in precursor lesions and early stages of bladder cancer.

Recent investigations have demonstrated alterations of the p53 tumor-suppressor gene in a considerable number of transitional-cell carcinoma (TCC) specimens. Thus far, these investigations have been restricted to either papillary TCC or invasive bladder cancer. To obtain further information on a possible involvement of p53 in bladder cancer development or tumor progression, investigations of precursor lesions and early stages of this disease are required. Immunohistochemical examination of 6 dysplasias and 24 carcinomas in situ (TIS) showed p53 accumulation, which is suggestive of p53 inactivation, in 2 (33%) and 9 (38%) of these specimens, respectively. This ratio was similar in 9 T1 lesions (33%) and in 14 cases of muscle-infiltrative disease (35%). In papillary tumors, p53 accumulation was observed exclusively in 3/10 moderately differentiated or high-grade lesions but not in 1 Ta G1 tumor. The expression of p53 accumulation was a consistent finding. The examination of tumor recurrences yielded either the presence or the absence of p53 overexpression in the primary and recurrent tumors of 7/8 patients. Similarly, in multifocal TCC, p53 accumulation was also either present or absent in 10/11 cases examined. These results suggest the existence of at least two different subgroups of TCC, with p53 accumulation being present in one of these groups. The observation of p53 accumulation in dysplasia and in TIS is a prerequisite for a possible involvement of p53 in bladder cancer carcinogenesis, although it does not prove this assumption.