Gert J. van Steenbrugge

Androgen-independent growth is induced by neuropeptides in human prostate cancer cell lines.

Androgen‐independent growth leads to progressive prostate cancer after androgen‐ablation therapy. This may be caused by altered specificity of the androgen receptor (AR), by ligand‐independent stimulation of the AR, or by paracrine growth modulation by neuropeptides secreted by neuroendocrine (NE) cells.

EXPRESSION OF MULTIDRUG RESISTANCE RELATED PROTEINS AND PROLIFERATIVE ACTIVITY IS INCREASED IN ADVANCED CLINICAL PROSTATE CANCER

PURPOSE Advanced disseminated prostate cancer is highly resistant to cytotoxic chemotherapy. We identified proteins that may be involved in multidrug resistance in clinical prostate cancer. Expression of these proteins was examined in the context of tumor progression. MATERIALS AND METHODS Paraffin embedded, formalin fixed prostate cancer specimens from archival sources of 3 distinct patient groups were examined. These groups were clearly distinct with regard to pathological stage and responsiveness to antihormonal therapy. Group 1 consisted of patients with organ confined prostate cancer treated with radical prostatectomy (early pathological stage T2N0M0). Group 2 patients had disseminated, early advanced prostate cancer and were treated with transurethral prostatic resection for urinary obstruction before receiving antihormonal therapy. Group 3 patients had disseminated prostate cancer with relapse despite antihormonal treatment (late advanced prostate cancer) and they underwent transurethral prostatic resection to relieve the symptoms of urinary obstruction. Immunohistochemical study was done to detect P-glycoprotein, multidrug resistance associated protein, lung resistance protein, glutathione-S-transferase pi, p53, Bcl-2, Bax, topoisomerase I, IIalpha and IIbeta, and Ki-67. RESULTS Advanced tumors were distinguished from locally confined tumors because they exhibited significantly higher histological grade and proliferative activity. The expression of multidrug resistance associated protein, p53, topoisomerase IIalpha, Ki-67 and topoisomerase IIbeta was significantly related to a higher Gleason sum score. The number of cases expressing multidrug resistance associated protein, lung resistance protein, glutathione-S-transferase pi, p53, Bcl-2, topoisomerase IIalpha and Ki-67 was significantly increased in the group with advanced disseminated prostate cancer. Topoisomerase I and IIbeta were homogeneously and highly expressed at all stages of prostate cancer progression, while P-glycoprotein was not expressed in any tumors regardless of the patient group. CONCLUSIONS Up-regulation of the expression of the drug transporters multidrug resistance associated protein and lung resistance protein, detoxifying enzyme glutathione-S-transferase pi, and apoptosis inhibiting proteins Bcl-2 and p53 may be an explanation of the resistance of disseminated progressive prostate cancer to chemotherapy. As shown by the up-regulation of Ki-67 and topoisomerase IIalpha, increased proliferation reflects the aggressiveness of metastatic prostate cancer. Research on agents that counteract multidrug resistance mechanisms and may sensitize prostate carcinoma to cytotoxic chemotherapy may possibly lead to more effective treatment of progressive disseminated prostate cancer.

Down‐regulation of CD44 expression in human prostatic carcinoma cell lines is correlated with DNA hypermethylation

Down‐regulation of the cell‐surface adhesion molecule CD44 has been suggested to play an important role in tumor progression and metastasis of prostate cancer. CD44 is encoded by a gene that contains a CpG‐rich region (CpG island) in its 5′ regulatory sequence. We tried to assess whether hypermethylation of this region is the mechanism responsible for CD44 transcriptional inactivation. A panel of prostatic‐carcinoma cell lines, Du145, LNCaP, PC3, PC346C and TSU, was analyzed for CD44 mRNA and protein expression. Du145, PC3 and TSU were positive for CD44, whereas in LNCaP and PC346C both CD44 mRNA and protein expression was suppressed. Methylation‐sensitive restriction‐enzyme analysis of genomic DNA showed that, in contrast to the CD44‐positive cell lines, the CD44‐negative lines were hypermethylated in the CD44 promoter CpG island. Furthermore, treatment of a PC346C culture with the demethylating agent 5‐azacytidine resulted in re‐expression of CD44 mRNA. It is concluded that hypermethylation of the CD44 5′ promoter region is one of the mechanisms by which CD44 expression is down‐regulated in prostatic‐carcinoma cell lines. Int. J. Cancer 80:439–443, 1999.

Neuroendocrine cells in the normal, hyperplastic and neoplastic prostate

Neuroendocrine cells can be demonstrated in normal, hyperplastic and neoplastic prostatic tissues. The products secreted by these cells can be used as tissue and/or serum markers but may also have biological effects. Neuroendocrine cells in prostate cancer most probably do not contain the androgen receptor and are therefore primarily androgen independent. Some of the neuropeptides secreted by the neuroendocrine cells may act as growth factor by activation of membrane receptors in an autocrine-paracrine fashion or by ligand-independent activation of the androgen receptor in neighboring non-neuroendocrine cells. Evidence is accumulating from experiments with tumor models that neuropeptides indeed can influence the growth of prostatic tumor cells. Future research on neuroendocrine differentiation may answer some questions concerning the biological behavior of clinical prostatic tumors.

Silencing of CD44 expression in prostate cancer by hypermethylation of the CD44 promoter region.

Loss of the CD44 transmembrane glycoprotein in primary prostate cancer has been shown to be associated with unfavorable clinical behavior. Moreover, the majority of prostate cancer metastases lack expression of this molecule. The mechanism of CD44 silencing in prostate cancer was investigated using both patient material and in vivo-propagated human prostate cancer xenografts. In 9 of 11 lymph node metastases of prostate cancer, we demonstrated by methylation-sensitive restriction enzyme digestion that the promoter region of the CD44 gene is methylated, indicating that this represents a major mechanism of CD44 silencing. Similarly, in 6 out of 12 in vivo-growing human prostate carcinoma xenograft models, hypermethylation of the CD44 gene was found. The extent of CpG island methylation was investigated by nucleotide sequencing after bisulphite modification of the CD44 promoter region. In the xenografts displaying hypermethylation, the examined 14 CpG sites in the CD44 transcription regulatory domain, including a Sp1 binding site, were consistently methylated. This correlated with reduced CD44 expression or lack of CD44 expression at mRNA and protein levels. In the xenografts lacking hypermethylation of the CD44 gene, high levels of CD44 mRNA and protein were expressed in some models, whereas in others CD44 mRNA expression was only detectable by RT-PCR and the CD44 protein could hardly be detected or was not detected at all. The results indicate that, in most prostate cancers, loss of CD44 expression is associated with extensive hypermethylation of the CpG island of the CD44 promoter region, but other, posttranscriptional mechanisms may also lead to CD44 loss.

Decreased expression of cd44 in metastatic prostate cancer

Decreased expression of CD44 is an independent prognostic marker for surgically treated prostate cancer. To investigate immunohistochemically defined CD44 expression in primary and metastatic prostate cancer, 2 groups of patients undergoing radical prostatectomy for clinically localized prostate cancer were studied. (1) pN1 group: 23 patients, finally staged pN1, of whom the radical prostatectomy specimen and the lymph nodes were investigated to establish a correlation between CD44 expression in the concurrently resected primaries and metastases; (2) pN0 group: 23 patients with pN0 disease matched for pT stage and Gleason sum score with the pN1 patients. Progression rates based on serum prostate‐specific antigen (PSA) levels could be determined in 42 of these 46 patients. In addition, 28 distant metastases were studied. A CD44 score of < 10% was found in 22 of the 23 lymph node metastases (96%) and in 20 of the corresponding radical prostatectomies. In the pN0 group this was observed in only 6 out of 23 specimens. In most of the distant metastases CD44 scores were < 10%. Patients with pN0 disease and > 10% CD44‐positive tumor cells had a significantly better prognosis than the other patients who were not significantly different from each other. CD44 expression is thus strongly reduced in prostate cancer metastases as well as in the corresponding primary tumors. This reduction may be used to predict the N stage clinically, provided that CD44 scores can be determined reliably on preoperative biopsy specimens. Int. J. Cancer (Pred. Oncol.) 84:478–483, 1999.

The cytocidal effect of high energy shock waves on human prostatic tumour cell lines

This report describes the effect of high energy shock waves (HESW) generated by a Siemens Lithostar on four human prostatic carcinoma cell lines in vitro. The effects of temperature, shock wave energy, cell density and the number of HESW were investigated. Pressure measurements were carried out in the focus of the lithotriptor and inside test tubes that were placed in the focus. Direct cell kill was inversely related to temperature, whereas a linear relationship was found with shock wave energy. Cell kill appeared to be independent of cell density. All four cell lines were sensitive to the treatment with HESW, but displayed a different dose-response pattern. In vitro treatment of PC-3 cells retarded their growth upon injection into nude mice. It is concluded that human prostatic tumour cells are killed by HESW. Therefore, HESW could be of potential value in tumour treatment.

THE PROGNOSTIC VALUE OF PRETREATMENT EXPRESSION OF ANDROGEN RECEPTOR AND BCL-2 IN HORMONALLY TREATED PROSTATE CANCER PATIENTS

PURPOSE We determined the prognostic value of oncoprotein bcl-2 and androgen receptor expression in pretreatment transurethral resection specimens of hormonally treated prostate cancer patients. MATERIALS AND METHODS A total of 68 pretreatment transurethral resection specimens, 30 radical prostatectomy specimens and 21 palliative transurethral resection specimens with androgen independent prostate cancer was stained with a monoclonal antibody against bcl-2. Androgen receptor immunohistochemistry was performed on pretreatment transurethral resection specimens only. Results were scored semiquantitatively and were correlated with tumor stage and grade and with the occurrence of clinical progression or tumor related death. RESULTS Bcl-2 expression by adenocarcinoma cells was found in 32, 17 and 24% of pretreatment transurethral resection, radical prostatectomy and palliative transurethral resection specimens, respectively. The bcl-2 scores did not correlate with tumor stage or grade. Androgen receptor was expressed in 88% of pretreatment transurethral resection specimens. Androgen receptor scores were marginally related to tumor grade, but not to tumor stage. A prognostic value of bcl-2 or androgen receptor in pretreatment transurethral resection specimens was not found. When a combined bcl-2/androgen receptor score was used, this parameter was an independent prognostic marker to predict clinical progression with Gleason grade and stage classification. Gleason grade was the only independent prognostic marker to predict tumor related death. CONCLUSIONS The expression of bcl-2 and androgen receptor in pretreatment prostate cancer specimens is not related to the prognosis of hormonally treated prostate cancer. Bcl-2 expression is not increased in endocrine therapy resistant prostate cancer. Surprisingly, a combined bcl-2/androgen receptor score acts as an independent prognosticator for clinical progression.

Decreased levels of topoisomerase IIα human renal cell carcinoma lines resistant to etoposide

Abstract Renal cell carcinoma (RCC) displays strong resistance against many chemotherapeutic drugs. Overexpression of P-glycoprotein (Pgp) appears to be part of this resistance. The involvement of another resistance mechanism, involving the decreased activity of DNA topoisomerase II (topoII), remains uncertain. By culturing the human RCC lines RC2 and RC21 in the presence of increasing concentrations of etoposide, we derived the variant sublines RC2E, RC21A and RC21E, that had acquired approximately 30-, 60- and 90-fold resistance to this drug respectively. RC2E, RC21A and RC21E were approximately 50-, 5- and 400-fold cross-resistant to doxorubicin respectively. RC2E and RC21E also showed cross-resistance (approximately 200- and 3500-fold respectively) to vinblastine. Quantitative differences in MDR1 and Pgp expression (elevated in RC2E and RC21E) and topoIIα (reduced in RC21E and RC21A) were demonstrated using Western blotting and the reverse transcriptase/polymerase chain reaction. Decreased amounts of topoIIα were reflected in a reduced activity of RC21A and RC21E as measured by unknotting phage P4 DNA. Qualitative changes of the topoIIα gene, such as point mutations in the motif B/DNBS and DNA-binding regions, or differences in methylation status of the promoter gene of RC21E, were not found. These cell lines represent a model of a solid tumor in which overexpression of Pgp, a combination of increased Pgp and decreased topoIIα, and a decrease of topoIIα are represented.

MIB-1 (KI-67) proliferation index and cyclin-dependent kinase inhibitor p27(Kip1) protein expression in nephroblastoma

Purpose: A number of studies have indicated that the tumor proliferation marker MIB-1 and cell cycle inhibitor p27Kip1 expression are of prognostic importance in a variety of cancers. The present study was performed to evaluate the prognostic value of these molecules in Wilms’ tumors. Experimental Design: MIB-1 and p27Kip1 expressions were investigated by the means of immunohistochemical analysis of 62 Wilms’ tumor. Patients were preoperatively treated by chemotherapeutic agents and had a mean follow-up of 5.7 years. Results: MIB-1 and p27Kip1 were expressed in normal kidney tissues and in the three main components of Wilms’ tumor, i.e., the blastemal, epithelial, and stromal cells. In Wilms’ tumors, the percentage of MIB-1-positive cells in the blastema ranged between 0 and 42% (mean, 9.4%) and in the epithelial component between 0 and 53% (mean, 19.9%), with a significant difference (P < 0.01). The percentage of blastemal p27Kip1-positive cells ranged between 3 and 85% (mean, 55.1%) and for the epithelial component between 1 and 87% (mean, 59%). There was a significant inverse relationship between blastemal MIB-1 and p27Kip1 expression in Wilms’ tumor. Univariate analysis showed that blastemal MIB-1 and p27Kip1 expression were indicative for clinical progression and tumor-specific survival. In a multivariate analysis, blastemal MIB-1 and p27Kip1 protein expression proved to be an independent prognostic for clinical progression besides stage. Conclusions: It was concluded that both MIB-1-based proliferative activity and p27Kip1 protein expression in the blastema have prognostic impact in Wilms’ tumor.