Heiner Renneberg

Molecular detection of thyroglobulin mRNA transcripts in peripheral blood of patients with thyroid disease by RT-PCR

The sensitive detection of circulating tumour cells in patients with differentiated thyroid cancer may precede the detection of relapse by other diagnostic studies – such as serum thyroglobulin – and thus may have important therapeutic and prognostic implications. We performed reverse transcription-polymerase chain reaction (RT-PCR) on blood samples from patients diagnosed with thyroid disease using two different RT-PCR sensitivities. Additionally, tissue specificity of TG mRNA-expression was determined using RNA extracts from 27 different human tissues. The lower limit of detection was 50–100 TG mRNA producing cells/ml blood using a ‘normal’ RT-PCR sensitivity and 10–20 cells/ml blood using a ‘high’ sensitivity. With the normal sensitivity TG mRNA was detected in 9/13 patients with thyroid cancer and metastasis, 63/137 patients with a history of thyroid cancer and no metastasis, 21/85 with non-malignant thyroid disease and 9/50 controls. With the high sensitivity TG mRNA was detected in 11/13 patients with thyroid cancer and metastasis, 111/137 patients with a history of thyroid cancer and no metastasis, 61/85 with non-malignant thyroid disease and 41/50 controls. Interestingly, using the normal RT-PCR sensitivity TG mRNA transcripts are specific for thyroid tissue and detectable in the peripheral blood of controls and patients with thyroid disease, which correlates with a diagnosis of metastasized thyroid cancer. However, with a high RT-PCR sensitivity, TG mRNA expression was found not to be specific for thyroid tissue and was not correlated with a diagnosis of thyroid cancer in patients. As a consequence, to date TG mRNA detected by RT-PCR in the peripheral blood cannot be recommended as a tumour marker superior to TG serum-level.

The significance of serum levels of insulin‐like growth factor‐1 in patients with prostate cancer

Objectives To compare the serum levels of insulin‐like growth factor‐1 (IGF‐1) in patients with prostate cancer and in control patients with no malignancy, and to evaluate any possible influence of testicular androgen withdrawal on the level of IGF‐1 in patients with prostate cancer.

Immunohistochemistry of prostasomes from human semen.

To elucidate the origin of so‐called “prostasomes” in human semen, a polyvalent rabbit antiserum was produced against a highly purified preparation of these secretory particles.

FIBRONECTIN IN HUMAN PROSTATIC CELLS IN VIVO AND IN VITRO : EXPRESSION, DISTRIBUTION, AND PATHOLOGICAL SIGNIFICANCE

Abstract In the present study we examined the expression and release of the extracellular matrix glycoprotein fibronectin (FN) in a prostate cancer cell line (LNCaP) and in primary prostatic stromal cells using the reverse transcription–polymerase chain reaction (RT-PCR) and by an enzyme-linked immunosorbent assay. Perturbation experiments in vitro using antibodies directed against FN and the FN receptor were also performed. Immunohistochemistry was used to show the in vivo distribution of FN and the FN receptor in tissue sections of normal human prostate, benign prostatic hyperplasia, and prostate carcinoma. The expression of the oncofetal FN ED-B segment in benign prostatic hyperplasia and prostate carcinoma tissue was investigated by RT-PCR. The FN mRNA was expressed by LNCaP and primary prostatic stromal cells, respectively. Both cell types released FN into the medium in a time-dependent manner, whereby FN secretion was about 2.5-fold higher in cultures of stromal cells relative to LNCaP cells. Blocking FN with anti-FN antibodies resulted in a significant decrease in cell adhesion for LNCaP cells and a change in morphology for the primary stromal cells. FN was located mainly in the stromal compartment of the prostate, showing a distinct distribution pattern in prostate carcinoma, whereas the FN receptor was detectable only in the prostate epithelia. RT-PCR experiments showed the expression of the oncofetal FN ED-B segment in benign prostatic hyperplasia and prostate carcinoma tissue, with a 3.5-fold higher expression in the prostate carcinoma probes. Our data point to an important role for FN in cell adhesion of prostatic cells and show that an alternatively spliced FN mRNA is upregulated in the pathologically altered human prostate.

Prognostic value of combined triple-reverse transcription-PCR analysis for prostate-specific antigen, human kallikrein 2, and prostate-specific membrane antigen mRNA in Peripheral blood and lymph nodes of prostate cancer patients

Purpose: We present the largest study of both peripheral blood and lymph node samples examining the utility of reverse transcription-polymerase chain reaction (RT-PCR) for established molecular markers as a diagnostic tool in the molecular staging of prostate cancer patients undergoing radical prostatectomy. Experimental Design: Peripheral blood from 358 patients was obtained before radical prostatectomy. Corresponding obturatory lymph node samples were collected from 153 of these patients. Nested RT-PCR for prostate-specific antigen (PSA), human kallikrein 2 (hK2), and prostate-specific membrane antigen (PSMA) were performed on cDNA from peripheral blood. The lymph node cDNA was analyzed for PSA und hK2 expression. Results: RT-PCR in peripheral blood was positive in 124 (34.6%) of 358 samples for PSA, 215 (60.1%) of 358 for PSMA, and 97 (27.1%) of 358 for hK2. Comparison of positive RT-PCR rates of pT2 and pT3 tumors in corresponding peripheral blood for PSA, PSMA, and hK2 were 31.9 and 40.0%, 58.8 and 62.5%, and 26.9 and 27.5%, respectively. Histopathologically, cancer-free lymph node samples were positive in RT-PCR for PSA and hK2 in 70 (49.6%) of 141 and 89 (63.2%) of 141 of cases. All histologically positive lymph node samples (n = 12, pN+) were positive for PSA RT-PCR. PSA RT-PCR alone, as well as combined PSA/PSMA RT-PCR evaluation, in peripheral blood showed a significant association with grading. PSA RT-PCR lymph node-negative samples were significantly less likely positive in their corresponding peripheral blood RT-PCR sample. Conclusions Although the preoperative PSA RT-PCR in peripheral blood correlated with the grading of prostate cancer, no combination of RT-PCR results using “triple” markers (PSA, hK2, PSMA) in peripheral blood and/or lymph nodes yielded additional preoperative staging information.

Cell lineage characteristics of human prostatic stromal cells cultured in vitro.

An in vitro model of prostatic stromal cells suitable for experimental studies of the pathogenesis of BPH is still lacking. We therefore standardized the isolation, cultivation, and characterization of human prostatic stromal cell lineages.

Transforming Growth Factor-β2 Mediates Mesenchymal-Epithelial Interactions of Testicular Somatic Cells12

Transforming growth factor-β2 (TGFβ2) is an important mediator of growth and differentiation. We here describe for the first time the complete sequence of the TGFβ2 complementary DNA derived from peritubular myoid cells of the rat testis. The size of the rat TGFβ2 complementary DNA was 1245 bp, and the deduced protein sequence contained 414 amino acids. Sequence comparison with the human and mouse amino acid sequences demonstrated 96.4% and 97.9% sequence identities, respectively. To elucidate the functional role of TGFβ2 in testicular somatic cells, we studied its secretion in vitro in monocultures and cocultures of mesenchymal peritubular and epithelial Sertoli cells. The highest amounts of TGFβ2 protein were secreted in the cocultures and by peritubular cells, whereas Sertoli cells secreted only minor amounts. Stimulation experiments with FSH revealed a reduced secretion of TGFβ2 in cocultures, probably mediated by a paracrine interaction of the FSH-responsive Sertoli cells. In contrast, TGFβ2 secretio...

Prostate specific membrane antigen (PSM) is expressed in various human tissues: implication for the use of PSM reverse transcription polymerase chain reaction to detect hematogenous prostate cancer spread

Abstract Detection of prostate-specific membrane antigen (PSM)-mRNA expression in blood samples using reverse transcription polymerase chain reaction (RT-PCR) is discussed as a new diagnostic marker of circulating micrometastases in prostate cancer patients. We applied the RT-PCR technique to different human tissues and obtained positive signals for PSM transcripts in human genital and multiple extra-genital tissue sites. The cDNAs were prepared from different human tissues and prostatic cell lines. RT-PCR and nested RT-PCR for PSM was performed with primers derived from the published PSM cDNA. The RT-PCR fragments obtained were cloned and showed 100% sequence homology to PSM. Southern blot hybridization with labeled probes was used to confirm the specificity of the amplicons. In addition to the known PSM expression in the human brain, PSM-mRNA was detected in cDNA isolated from human testis, epididymis and seminal vesicles and in the PC-3 prostatic cancer cell line. Furthermore, we found PSM-mRNA in heart, liver, lung, kidney, spleen, and thyroid gland. The results indicate that PSM expression is not restricted to the prostate gland, but represents a more general component of genital and extra-genital human tissues. This must be considered when RT-PCR and nested RT-PCR screening for PSM expression is performed as a diagnostic measure in blood from prostate cancer patients.

Ribozyme-targeting of a secreted FGF-binding protein (FGF-BP) inhibits proliferation of prostate cancer cells in vitro and in vivo

Prostate cancer is one of the most common malignant tumors with increasing incidence rates in the aging male. Since locally advanced or metastatic prostate tumors are essentially incurable, identification of new target molecules and treatment strategies is of critical importance. Fibroblast growth factor-2 (FGF-2) acts as potent mitogen which is upregulated in prostate cancers modulating cancer cell proliferation and development of an invasive phenotype. Normally it is tightly bound to the extracellular matrix that quenches its biological activity. The FGF-binding proteins (FGF-BP, HBp17) is a secreted protein which is able to mobilize and activate FGF-2 from the extracellular matrix. Here we show that FGF-BP is highly expressed in prostate tumor cells. To study the functional role of FGF-BP, we use a ribozyme-targeting approach to selectively deplete FGF-BP in prostate cancer cells achieving a more than 50% reduction of FGF-BP mRNA and protein levels in two mass-transfected cell lines. FGF-BP depletion reduces proliferation of the cells in vitro without changes in cell cycle distribution or apoptosis. Using cDNA microarrays, Northern blotting and RT–PCR, we show a complex pattern of changes in the gene expression profiles upon FGF-BP depletion. Most strikingly, ribozyme-mediated reduction of FGF-BP levels completely abolishes the ability of the highly metastatic PC-3 prostate carcinoma cells to grow tumors in an athymic nude mouse in vivo model which is far beyond the effects of FGF-BP ribozyme targeting observed previously in cells from other tumors in the same model. Taken together, our study identifies FGF-BP as a potential rate-limiting factor for prostate cancer growth and, due to its restricted expression pattern in adults, a potentially attractive target for prostate cancer therapy.

Semiquantitative morphology of human prostatic development and regional distribution of prostatic neuroendocrine cells

The neuroendocrine cells of the human prostate have been related to proliferative disorders such as prostatic cancer. Their origin, distribution, and development have therefore been studied and discussed in terms of current stem cell concepts in the prostate.