Neoplastic Prostate Research Articles

Skeletal metastasis of prostate adenocarcinoma in rats: Morphometric analysis and role of parathyroid hormone‐related protein

BACKGROUND Prostate cancer frequently metastasizes to bone, where it induces osteoblastic lesions. Parathyroid hormone-related protein (PTHrP), a product of normal and neoplastic prostate cells, may promote growth and bone metastasis of certain types of cancer. In this study, we investigated the: 1) pathogenesis and morphology of bone metastases in the MATLyLu rat prostate adenocarcinoma model, and 2) effect of PTHrP overexpression on tumor growth and incidence of bone metastasis. METHODS MATLyLu cells were stably transfected with a PTHrP expression vector or control plasmid. PTHrP expression was determined in vitro by immunoradiometric assay and Northern blot analysis. MATLyLu cells were injected into the left ventricle of Copenhagen rats to induce bone metastases. Histology and radiography were used to quantify the size and number of bone metastases. Serum alkaline phosphatase isoenzyme concentrations and histomorphometric analysis were used to evaluate bone formation and resorption. RESULTS All rats developed osteolytic metastases in long bones and vertebrae. There was no evidence of increased intramedullary bone formation. PTHrP overexpression by MATLyLu cells was not associated with any difference in the incidence of bone metastasis, size of metastatic foci or tumor-cell proliferation. CONCLUSIONS The MATLyLu intracardiac injection model of prostate carcinoma is an aggressive tumor model with a high incidence of osteolytic skeletal metastases, and is not altered by increased PTHrP production by neoplastic prostate epithelial cells. Prostate 39:187–197, 1999. © 1999 Wiley-Liss, Inc.

Skeletal metastasis of prostate adenocarcinoma in rats: morphometric analysis and role of parathyroid hormone-related protein.

Prostate cancer frequently metastasizes to bone, where it induces osteoblastic lesions. Parathyroid hormone-related protein (PTHrP), a product of normal and neoplastic prostate cells, may promote growth and bone metastasis of certain types of cancer. In this study, we investigated the: 1) pathogenesis and morphology of bone metastases in the MATLyLu rat prostate adenocarcinoma model, and 2) effect of PTHrP overexpression on tumor growth and incidence of bone metastasis. MATLyLu cells were stably transfected with a PTHrP expression vector or control plasmid. PTHrP expression was determined in vitro by immunoradiometric assay and Northern blot analysis. MATLyLu cells were injected into the left ventricle of Copenhagen rats to induce bone metastases. Histology and radiography were used to quantify the size and number of bone metastases. Serum alkaline phosphatase isoenzyme concentrations and histomorphometric analysis were used to evaluate bone formation and resorption. All rats developed osteolytic metastases in long bones and vertebrae. There was no evidence of increased intramedullary bone formation. PTHrP overexpression by MATLyLu cells was not associated with any difference in the incidence of bone metastasis, size of metastatic foci or tumor-cell proliferation. The MATLyLu intracardiac injection model of prostate carcinoma is an aggressive tumor model with a high incidence of osteolytic skeletal metastases, and is not altered by increased PTHrP production by neoplastic prostate epithelial cells.

Distribution of prostasomes in neoplastic epithelial prostate cells.

Prostasomes are a secretory product from the prostate. We aimed to investigate whether the distribution and amount of prostasomes in normal prostate epithelium were influenced by the dedifferentiation occurring in adenocarcinomas of the human prostate gland. Transurethrally resected material from 11 patients with prostatic carcinoma of various malignancy grades, material from two lymph node metastases, and benign tissue from 10 total prostatectomies were subjected to immunohistochemical staining, using a mouse monoclonal antibody against human prostasomes (mAb78). Immunostaining of low-grade carcinoma was similar to that of normal prostate gland which displayed a cytoplasmic granular staining of the apical (luminal) aspects of the secretory epithelial cells. In moderately well and poorly differentiated adenocarcinoma, the amount of stained components decreased, and the staining pattern became more heterogeneous. In multilayered glandular structures, the staining was concentrated at the lumen, leaving most other cells negative. The neoplastic cells of lymph node metastases of prostate carcinoma differed in amount and distribution of immunostained prostasomes. The antigen recognized in the prostasomes by mAb78 was expressed in benign prostate tissue, prostate cancer, and to a lesser degree in lymph node metastases. There was a tendency towards decreased expression with increasing tumor grade.

Molecular cloning and characterization of prostase, an androgen-regulated serine protease with prostate-restricted expression

The identification of genes with selective expression in specific organs or cell types provides an entry point for understanding biological processes that occur uniquely within a particular tissue. Using a subtraction approach designed to identify genes preferentially expressed in specific tissues, we have identified prostase, a human serine protease with prostate-restricted expression. The prostase cDNA encodes a putative 254-aa polypeptide with a conserved serine protease catalytic triad and an amino-terminal pre-propeptide sequence, indicating a potential secretory function. The genomic sequence comprises five exons and four introns and contains multiple copies of a chromosome 19q-specific minisatellite repeat. Northern analysis indicates that prostase mRNA is expressed in hormonally responsive normal and neoplastic prostate epithelial tissues, but not in prostate stromal constituents. Prostase shares 35% amino acid identity with prostate-specific antigen (PSA) and 78% identity with the porcine enamel matrix serine proteinase 1, an enzyme involved in enamel matrix degradation and with a putative role in the disruption of intercellular junctions. Radiation-hybrid-panel mapping localized prostase to chromosome 19q13, a region containing several other serine proteases, including protease M, pancreatic/renal kallikrein hK1, and the prostate-specific kallikreins hK2 and hK3 (PSA). The sequence homology between prostase and other well-characterized serine proteases suggests several potential functional roles for the prostase protein that include the degradation of extracellular matrix and the activation of PSA and other proteases.

A Critical Evaluation of Prostate Specific Antigen Parameter Responses to Manipulation According to Prostatic Histology

Introduction: The degree of increase in prostate specific antigen (PSA) forms after prostatic manipulation may differ depending on histology. We sought to critically evaluate this hypothesis and to examine possible clinical implications. Methods: A series of 198 men referred for transrectal ultrasound‐guided biopsy on clinical grounds had free (fPSA) and total PSA (tPSA) levels determined before undergoing biopsy and 30 min after biopsy. A subset of 100 men had the same analysis immediately before and 30 min after digital rectal examination (DRE). Geometric mean ratios and two‐sample t tests compared the change in levels of fPSA, PSA, the ratio of fPSA to tPSA (f/t PSA), fPSA density, and PSA density after manipulation to prostatic histology. Results: After undergoing biopsies, 57 men (29%) received a diagnosis of prostate cancer. The biopsy significantly altered all parameters. The degree of change was less in the malignant group for all parameters, although this only approached statistical significance for PSA and PSA density levles (p = 0.159 and p = 0.170, respectively). In the DRE group, the PSA changes were similar, although the cancer patients tended to release slightly less PSA forms (PSA, p = 0.376; fPSA, p = 0.657; f/t PSA, p = 0.396). Conclusion: PSA release and PSA parameter alteration after manipulation differ in the neoplastic prostate. The subtlety of change after DRE currently limits clinical application.

Impaired expression and posttranslational processing of connexin43 and downregulation of gap junctional communication in neoplastic human prostate cells.

Gap junctional communication (GJC) has been implicated in the control of cell proliferation. Numerous cancer cells show a decrease or loss of GJC compared to their normal counterparts. Lack of adequate information on the status of gap junctions during prostate neoplasia prompted us to examine this form of cancer, which comprises about 14% of male cancer deaths in America. Cultured normal human prostate epithelial cells and several different human prostate tumor lines were used in this study. GJC was assayed by dye transfer, whereas Western blot and immunofluorescence methods were used to examine connexin43 (Cx43) levels and the presence of gap junctions, respectively. Normal human prostate cultures exhibited extensive cell-communication which was completely absent in all the examined tumor cells. This disrupted communication was associated with a decreased expression and an impaired posttranslational modification of Cx43 in these cells. Abundant immunostaining of gap junctional channels by a Cx43-antibody was observed in normal prostate cells but not in tumor cells. Our data provide further support for the hypothesis that loss of junctional communication is a critical step in progression to human prostate neoplasia.

Codistribution of procathepsin B and mature cathepsin B forms in human prostate tumors detected by confocal and immunofluorescence microscopy

Cathepsin B (CB) is involved in invasion and metastasis of a variety of solid organ tumors, including human prostate cancer. The tertiary structures of the proenzyme and mature forms of CB are related closely, as revealed by crystallographic studies. However, the cellular distributions of the CB forms have not been defined in human prostate and its tumors. Our objective was to investigate the distribution and codistribution of CB and procathepsin B (proCB) in human prostate tumors. Human prostate tissue samples that were obtained from 21 prostatectomy and/or cystectomy patients were collected immediately after surgery and processed for this study. We used a rabbit antihuman liver CB immunoglobulin G (IgG) that recognizes both mature CB and proCB and a mouse antipropeptide monoclonal antibody IgG that recognizes only proCB. Fluorescein isothiocyanate (FITC)-conjugated donkey antirabbit IgG and indocarbocyanine (Cy3; rhodamine)-conjugated donkey antimouse IgG were used to differentiate localization of the enzyme forms. Immunofluorescence of FITC and Cy3 was examined in prostate sections by using epifluorescence and confocal laser-scanning microscopy. Because fluorescence is dependent on section thickness, time needed for study and photography, and the antigenic sites of proCB and mature CB localized by antibodies and by fluorescent markers (Cy3 vs. FITC), the cellular distributions and the relative intensity of fluorescence on cryostat sections were assessed qualitatively. Immunofluorescence of Cy3 for localizing proCB and of FITC for localizing mature CB were observed in prostatic epithelial cells and their tumors and in stromal connective tissue cells. By using confocal microscopy, colocalization of the enzyme forms in the same cells was indicated by yellow fluorescence. In stromal cells (such as smooth muscles, fibroblast, and macrophages), the distribution of proCB and relative fluorescence intensity was moderate to predominant in human prostate and its tumors. In neoplastic prostate, the cellular distributions of CB ranged from low to predominant levels. In some neoplastic glands, Cy3 fluorescence for proCB was absent, whereas the mature form of CB localized in cancer cells and in the subjacent extracellular matrix. Confocal microscopy showed a close association of CB with extracellular matrix surrounding neoplastic acini and invasive cells, indicating that the enzyme form was probably involved in degradation of the matrix proteins. The negative control study showed no specific immunofluorescence for proCB or CB in prostate cancer cases. We have shown a differential distribution of proenzyme and mature forms of CB in normal prostate, benign prostatic hyperplasia, and neoplastic prostate. The enzyme forms were assessed by determining the cellular distributions of CB and proCB. Our study indicates that the differential distribution of proCB and CB might provide clues into aggressiveness of prostate cancers within Gleason grades. However, we emphasize that our observation should be evaluated in a larger series of prostate samples before a definitive conclusion can be reached. This is the first report to show codistribution of proenzyme and mature forms of CB by using confocal microscopy. Anat. Rec. 252:281–289, 1998. © 1998 Wiley-Liss, Inc.

Codistribution of procathepsin B and mature cathepsin B forms in human prostate tumors detected by confocal and immunofluorescence microscopy.

Cathepsin B (CB) is involved in invasion and metastasis of a variety of solid organ tumors, including human prostate cancer. The tertiary structures of the proenzyme and mature forms of CB are related closely, as revealed by crystallographic studies. However, the cellular distributions of the CB forms have not been defined in human prostate and its tumors. Our objective was to investigate the distribution and codistribution of CB and procathepsin B (proCB) in human prostate tumors. Human prostate tissue samples that were obtained from 21 prostatectomy and/or cystectomy patients were collected immediately after surgery and processed for this study. We used a rabbit antihuman liver CB immunoglobulin G (IgG) that recognizes both mature CB and proCB and a mouse antipropeptide monoclonal antibody IgG that recognizes only proCB. Fluorescein isothiocyanate (FITC)-conjugated donkey antirabbit IgG and indocarbocyanine (Cy3; rhodamine)-conjugated donkey antimouse IgG were used to differentiate localization of the enzyme forms. Immunofluorescence of FITC and Cy3 was examined in prostate sections by using epifluorescence and confocal laser-scanning microscopy. Because fluorescence is dependent on section thickness, time needed for study and photography, and the antigenic sites of proCB and mature CB localized by antibodies and by fluorescent markers (Cy3 vs. FITC), the cellular distributions and the relative intensity of fluorescence on cryostat sections were assessed qualitatively. Immunofluorescence of Cy3 for localizing proCB and of FITC for localizing mature CB were observed in prostatic epithelial cells and their tumors and in stromal connective tissue cells. By using confocal microscopy, colocalization of the enzyme forms in the same cells was indicated by yellow fluorescence. In stromal cells (such as smooth muscles, fibroblast, and macrophages), the distribution of proCB and relative fluorescence intensity was moderate to predominant in human prostate and its tumors. In neoplastic prostate, the cellular distributions of CB ranged from low to predominant levels. In some neoplastic glands, Cy3 fluorescence for proCB was absent, whereas the mature form of CB localized in cancer cells and in the subjacent extracellular matrix. Confocal microscopy showed a close association of CB with extracellular matrix surrounding neoplastic acini and invasive cells, indicating that the enzyme form was probably involved in degradation of the matrix proteins. The negative control study showed no specific immunofluorescence for proCB or CB in prostate cancer cases. We have shown a differential distribution of proenzyme and mature forms of CB in normal prostate, benign prostatic hyperplasia, and neoplastic prostate. The enzyme forms were assessed by determining the cellular distributions of CB and proCB. Our study indicates that the differential distribution of proCB and CB might provide clues into aggressiveness of prostate cancers within Gleason grades. However, we emphasize that our observation should be evaluated in a larger series of prostate samples before a definitive conclusion can be reached. This is the first report to show codistribution of proenzyme and mature forms of CB by using confocal microscopy.

PAGE-1, an X chromosome-linked GAGE-like gene that is expressed in normal and neoplastic prostate, testis, and uterus.

We have used a combination of computerized database mining and experimental expression analyses to identify a gene that is preferentially expressed in normal male and female reproductive tissues, prostate, testis, fallopian tube, uterus, and placenta, as well as in prostate cancer, testicular cancer, and uterine cancer. This gene is located on the human X chromosome, and it is homologous to a family of genes encoding GAGE-like proteins. GAGE proteins are expressed in a variety of tumors and in testis. We designate the novel gene PAGE-1 because the expression pattern in the Cancer Genome Anatomy Project libraries indicates that it is predominantly expressed in normal and neoplastic prostate. Further database analysis indicates the presence of other genes with high homology to PAGE-1, which were found in cDNA libraries derived from testis, pooled libraries (with testis), and in a germ cell tumor library. The expression of PAGE-1 in normal and malignant prostate, testicular, and uterine tissues makes it a possible target for the diagnosis and possibly for the vaccine-based therapy of neoplasms of prostate, testis, and uterus.

Effect of sesamin on growth and arachidonic acid content of neoplastic and non-neoplastic prostate epithelial cell cultures

Sesamin (a lignan present in sesame oil) is reported to specifically inhibit Δ5 desaturation and the formation of arachidonic acid (20:4n-6) from dihomogamma-linolenic acid (20:3n-6) in cell free extracts. In this study the effects of sesamin have been evaluated on cell cultures derived from canine prostatic tissues of epithelial origin from neoplastic and non-neoplastic tissues. Sesamin reduced cell proliferation in a dose dependent manner in both cell lines with TD 50 values of 91 ± 16 μM (non-neoplastic) and 132 ± 12 μM (neoplastic). In the non-neoplastic cells, supplemented with gamma-linolenic acid (18:3n-6) in the culture media, sesamin had little effect on the content of C16 and C18 fatty acids but markedly reduced the content of 20:4n-6 and caused an accumulation of 20:3n-6. This observation is consistent with sesamin inhibiting Δ5' desaturase activity. Sesamin, however, had little effect on the content and ratio of 20:3n-6 and 20:4n-6 fatty acids in neoplastic cells indicating that Δ5 desaturase activity was low in these cells. Thus, sesamin is an effective compound for reducing Δ5 desaturase activity in non-neoplastic cell cultures and could be used to manipulate the content of series 1 and 2 prostaglandins derived from 20:3n-6 and 20:4n-6, respectively.

Prostatectomy tissue for research: balancing patient care and discovery.

The application of modern technology in basic research often requires fresh tissue from human organs. The acquisition of this tissue challenges the anatomic pathologist to balance the needs of the basic scientist with the requirements of quality patient care. Our experience indicates that fresh tissue adequate for research can be obtained from neoplastic prostate glands without compromising patient care. The process requires dedicated, knowledgeable individuals and extensive documentation. Potential problems include low yield for research studies, loss of all residual carcinoma, compromise of the specimen margin, and unforeseen costs. Best collaborations occur in situations in which basic scientists and anatomic pathologists establish a working relationship and develop a mutually supportive means of funding.

Expression of periglandular tenascin-C and basement membrane laminin in normal prostate, benign prostatic hyperplasia and prostate carcinoma.

To evaluate the structural relationship of the distribution between tenascin (tenascin-C, an extra-cellular matrix glycoprotein involved in stromal-epithelial interactions in both normal and pathological conditions) and laminin, an important component of the basement membrane, in normal and neoplastic human prostate, and to establish whether changes in the basement membrane are accompanied by changes in tenascin staining. Seventy-five snap-frozen prostate samples representing normal glands, nodular benign prostatic hyperplasia and prostate carcinoma were stained for tenascin. From these, 15 samples were selected for dual-immunofluorescence staining and a confocal laser scan microscope was used to simultaneously visualize tenascin and laminin immunoreactivity. Tenascin was expressed in the extracellular matrix, mainly at the periphery of the glands, in tumour foci and blood vessels. In cases with intact basement membranes, e.g. normal glands and hyperplastic lesions, tenascin expression was weak. Low- and moderate-grade tumours were characterized by strong tenascin expression, while laminin expression was weak and/or showed discontinuities, indicating disturbances in basement membrane composition. High-grade tumours had sparse tenascin staining and a marked loss of laminin immunoreactivity. These results indicate that periglandular tenascin expression correlates with the integrity of the basement membrane in the human prostate. By influencing stromal-epithelial interactions, tenascin may play a role in maintaining tissue homeostasis in the prostate.

Synthetic matrix metalloproteinase inhibitor, BB-94, inhibits the invasion of neoplastic human prostate cells in a mouse model.

It has been suggested that increased metalloproteinase activity is a critical event in neoplastic progression leading to the initiation of local invasion and ultimately to the dissemination of neoplastic cells. This has led to an interest in testing the ability of metalloproteinase inhibitors to prevent the progression of carcinoma in situ into invasive and, therefore, more malignant tumors. One such agent is the synthetic matrix metalloproteinase inhibitor, BB-94. The effect of BB-94 on the intrinsic invasive potential of matrilysin-transfected Du-145 cells was evaluated by an in vitro invasion assay. In addition a diaphragm invasion model, which provides an easily oriented structure in which the earliest penetration of the basal lamina can be observed, was used to investigate the effect of BB-94 on the invasion and growth of tumors formed by these cells when injected into S.C.I.D. mice. The synthetic matrix metalloproteinase inhibitor, BB-94, was shown to effectively inhibit the invasion of matrigel and murine diaphragm. Metalloproteinase inhibitors, such as BB-94, that are able to limit tumor growth, and local invasion, may decrease the invasion of invasive carcinomas.

QUANTITATIVE IMMUNOHISTOCHEMICAL ANALYSIS OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 IN HUMAN PROSTATIC ADENOCARCINOMA: A PROGNOSTIC STUDY

Purpose We sought to characterize and quantitate the expression of IGFBP-3 in adenocarcinoma of the prostate and to test whether it correlated with tumor differentiation determined by Gleason grade. We also investigated the potential of using IGFBP-3 as a prognostic indicator of clinically localized prostate cancer. Materials and Methods Initially we evaluated the expression of IGFBP-3 in six normal and twenty neoplastic prostates using standard immunohistochemical techniques (study 1). We then obtained radical prostatectomy specimens from twenty-four patients with a preoperative diagnosis of clinically localized prostate adenocarcinoma and five year follow up information, and nine normal prostates from organ donors or from patients undergoing cystoprostatectomy (study 2). All specimens were immunostained with a polyclonal anti-human IGFBP-3 antibody. A single pathologist reviewed all sections and assigned a Gleason grade to each cancer focus. Using computer-assisted video image analysis, we quantified the intensity of IGFBP-3 immunostaining of each cancer focus and of normal controls. Results Normal prostatic epithelium showed intense cytoplasmic IGFBP-3 staining. The stromal compartment showed less intense staining, although there were occasional areas with strong immunoreactivity. The cellular distribution of IGFBP-3 staining in prostatic adenocarcinoma was comparable to normal tissue; however, the intensity of detectable staining in neoplastic epithelial cells was significantly decreased. Two foci of prostatic intraepithelial neoplasia (PIN) demonstrated IGFBP-3 immunoreactivity decreased in comparison to normal epithelium, but greater than prostatic adenocarcinoma. Histologically normal epithelium surrounding cancer foci also showed decreased immunostaining for IGFBP-3 compared with normal prostate. The marked decrease in immunostaining intensity of IGFBP-3 in prostate adenocarcinoma was not associated with Gleason grade or with clinical outcome. Conclusion Malignant transformation of prostatic epithelium was associated with a significant decrease in the amount of immunoreactive IGFBP-3 (p <0.0001); however, this parameter did not correlate with Gleason grade of the tumor or with patient outcome. The decrease in immunostaining intensity of IGFBP-3 in all Gleason grades and in PIN suggests that lower expression of IGFBP-3 is an early event in prostatic carcinogenesis. The finding that decreased IGFBP-3 immunostaining did not correlate with clinical outcome suggests that this parameter is not a therapy-guiding prognostic indicator for clinically localized prostate cancer.

Characterization of the cell-specific expression of parathyroid hormone–related protein in normal and neoplastic prostate tissue

Objectives. Parathyroid hormone–related protein (PTHrP) is a primary factor in the pathogenesis of malignancy-associated hypercalcemia. By alternative splicing, the human PTHrP gene can generate three different species of mRNA that encode three initial translational isoforms of 139, 173, and 141 amino acids. We recently reported that PTHrP was present in normal prostatic neuroendocrine cells and was overexpressed in prostate cancer tissue as demonstrated by immunostaining. This study was undertaken to further clarify the complex expression of PTHrP gene in normal prostate tissue and prostate cancer. Methods. PTHrP mRNA in samples prepared from normal prostate tissue, prostate cancer, and three prostate cancer cell lines, PC3, LNCaP, and DU145 was assessed using Northern hybridization. Expressed PTHrP isoforms were deduced from differential reverse transcription-polymerase chain reaction (RT-PCR) assays with exon-specific primers. Further localization of different species of PTHrP mRNA was performed using nonradioactive in situ hybridization with exon-specific probes on consecutive sections of normal and neoplastic prostate tissue. Results. Northern hybridization showed that the PTHrP expression level was higher in prostate cancer than in normal prostate tissue. All three PTHrP isoforms could be detected in normal prostate tissues and prostate cancer with differential RT-PCR. Further analysis using in situ hybridization with exon-specific probes revealed that all three PTHrP isoforms were present in prostatic neuroendocrine cells and only PTHrP-1–139 isoform could be clearly detected in prostate cancer tissue. Two androgen-insensitive cell lines, PC3 and DU145, derived from a bone metastasis and a brain metastasis, respectively, expressed all three mRNA species encoding for the three isoforms, but DU145 cells expressed less than PC3 cells. Androgen-sensitive LNCaP cells exhibited a low level of expression of mRNA species encoding for PTHrP-1–139 and PTHrP-1–173, and no expression of PTHrP1-141 isoform. Conclusions. All three initial translational isoforms of PTHrP are produced by prostatic neuroendocrine cells. The mature products of PTHrP might exert their effects on other prostatic epithelial cells in a paracrine fashion and also participate in the homeostatic regulation of the ejaculate. In prostate cancer, differential expression of these three isoforms is evident and PTHrP-1–139 isoform is more abundant than the other two forms. These findings are valuable for designing future research studies to further elucidate the biological functions of PTHrP in normal prostatic glands and prostate cancer.

Altered expression of RET proto-oncogene product in prostatic intraepithelial neoplasia and prostate cancer.

The RET proto-oncogene encodes a protein that belongs to the tyrosine kinase growth factor receptor family. Germline point mutations in RET are found in individuals with multiple endocrine neoplasia (MEN) syndromes, and gene rearrangements have been reported in papillary thyroid cancers. We recently identified transcripts of the RET proto-oncogene in human prostate cancer xenografts and prostate cancer cell lines by means of reverse transcription-polymerase chain reaction analyses. The purpose of this study was to investigate Ret protein expression in human prostate tissue. Ret protein expression was evaluated immunohistochemically in formalin-fixed, paraffin-embedded whole-prostate sections. The prostate specimens were obtained from 30 patients with prostate cancer after radical prostatectomies. Ret protein expression was compared in tumor foci and benign prostatic tissue. Medullary thyroid carcinoma tissue associated with an MEN syndrome and papillary thyroid cancer tissue served as positive controls. Ret appeared to be overexpressed in high-grade (histopathologically advanced) prostatic intraepithelial neoplasia (PIN) and prostate cancer when compared with its expression level in benign prostatic secretory epithelium. In addition, there was an apparent increase in Ret protein expression with decreased cellular differentiation, i.e., increasing Gleason pattern. Expression of the RET proto-oncogene in benign prostatic epithelium, high-grade PIN, and histopathologically advanced prostate cancer suggests that RET may play a role in the growth of both benign and neoplastic prostate epithelial cells.

DIFFERENTIAL EXPRESSION OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEINS IN HIGH VERSUS LOW GLEASON SCORE PROSTATE CANCER

Purpose The insulin-like growth factor (IGF) system appears to be important in human prostate cancer biology. Expression of specific IGF binding proteins (IGFBPs) by prostate cancer tissues may modulate IGF cellular actions, and possibly determine both IGF-dependent tumor growth and biological aggressiveness in vivo. The purpose of this study was to examine the differential expression of all six IGFBP genes in benign and malignant prostate tissue samples. Materials and Methods Using RNAse protection assays, we examined expression of IGFBPs 1 through 6 in 23 paired benign and neoplastic prostate tissue samples obtained from the same prostatectomy specimen. RNA expression levels on each tissue sample were determined densitometrically and groups compared using standard Student's t test. Results We found expression of IGFBPs 2 through 6, but not IGFBP-1, in both malignant and benign tissues. A statistically significant differential expression of IGFBPs 2, 3 and 5 was found between tumors with high Gleason score and those with low scores and benign tissues. Expression of IGFBPs 2 and 5 was higher (p = 0.002 and 0.04, respectively) while that of IGFBP-3 was lower (p = 0.05) in high versus low Gleason score cancer specimens. Expression of IGFBPs 4 and 6 was no different between tumors (p = 0.052 and 0.25, respectively). No significant differences in IGFBP expression were evident between benign and tumor tissues when tumor grade was not considered. Conclusion Our results indicate that differential expression of certain IGFBPs in human prostate cancer correlates with tumor Gleason score. Thus, expression of certain IGFBPs in prostate cancer may be used as a surrogate marker of aggressive clinical behavior.

Expression of KAI1 in paraffin-embedded normal, hyperplastic and neoplastic prostate and prostate carcinoma cell lines.

Expression of KAI1, a tumor metastasis suppressor gene, was studied with different fixatives in frozen and paraffin-embedded sections of human and rat prostate carcinoma cell lines and human prostate lesions by immunohistochemistry. Immunoreactivity of the membrane antigen in cell lines was associated with known expression levels in these lines and the fixative used. Formalin and paraformaldehyde helped maintain the immunoreactivity of cells. In human prostate, frozen sections revealed diffuse reactivity of the antigen in normal and neoplastic tissues while paraffin-embedded tissues usually showed focal reactivity, although more than 50% of cases with normal epithelium and adenocarcinomas were reactive. In some cases, pretreatment with trypsin enhanced immunoreactivity. Benign prostatic hyperplasia (BPH) showed the most intense diffuse immunoreactivity, which suggested enhanced expression. Prostatic intraepithelial neoplasia (PIN) also often expressed high levels of KAI1. Three of five metastases were reactive but two primaries and their metastases were not. Lymphocytes in primary carcinomas and lymphocytes and germinal center cells in lymph nodes were immunoreactive, while adjacent primary or metastatic prostate adenocarcinoma epithelium was not immunoreactive. Although paraffin-embedded human tissues were not optimal for determining levels of expression of KAI1, they did show immunoreactivity that could have prognostic value and showed the specific cytoplasmic localization of the protein in cells.

Expression of Granulocyte-Macrophage Colony-Stimulating Factor Receptors in Human Prostate Cancer

We studied the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in the human prostate carcinoma cell line LNCaP and looked for its presence in normal and neoplastic human prostatic tissue. The GM-CSF receptor is composed of two subunits, alpha and beta. While the isolated alpha subunit binds GM-CSF at low-affinity, the isolated beta subunit does not bind GM-CSF by itself; but complexes with the alpha subunit to form a high-affinity receptor. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed expression of mRNAs encoding the alpha and beta subunits of the GM-CSF receptor in LNCaP cells, and the presence of the alpha and beta proteins was confirmed by immunolocalization with anti-alpha and anti-beta antibodies. Receptor binding studies using radiolabeled GM-CSF showed that LNCaP cells have about 150 high-affinity sites with a kd of 40 pmol/L and approximately 750 low-affinity sites with a kd of 2 nmol/L. GM-CSF signaled, in a time- and dose-dependent manner, for protein tyrosine phosphorylation and induced the proliferation of the LNCaP cells. Immunolocalization studies showed low level expression of GM-CSF alpha and beta subunits in normal prostate tissue, with substantial expression in benign prostatic hyperplasia and prominent expression in neoplastic prostate tissue. Maximal expression of both subunits was observed in prostatic carcinomas metastatic to lymph node and bone. Tumor cells that stained positively with anti-alpha subunit antibodies were also reactive with anti-beta subunit antibodies, indicating that they express high-affinity GM-CSF receptors. Our data show that the LNCaP cells express functional GM-CSF receptors and that prostatic carcinomas have prominent GM-CSF receptor expression. These findings imply that both hyperplastic and neoplastic prostatic tissues may be responsive to GM-CSF.

Expression of Granulocyte-Macrophage Colony-Stimulating Factor Receptors in Human Prostate Cancer

AbstractWe studied the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor in the human prostate carcinoma cell line LNCaP and looked for its presence in normal and neoplastic human prostatic tissue. The GM-CSF receptor is composed of two subunits, α and β. While the isolated α subunit binds GM-CSF at low-affinity, the isolated β subunit does not bind GM-CSF by itself; but complexes with the α subunit to form a high-affinity receptor. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed expression of mRNAs encoding the α and β subunits of the GM-CSF receptor in LNCaP cells, and the presence of the α and β proteins was confirmed by immunolocalization with anti-α and anti-β antibodies. Receptor binding studies using radiolabeled GM-CSF showed that LNCaP cells have about 150 high-affinity sites with a kd of 40 pmol/L and approximately 750 low-affinity sites with a kd of 2 nmol/L. GM-CSF signaled, in a time- and dose-dependent manner, for protein tyrosine phosphorylation and induced the proliferation of the LNCaP cells. Immunolocalization studies showed low level expression of GM-CSF α and β subunits in normal prostate tissue, with substantial expression in benign prostatic hyperplasia and prominent expression in neoplastic prostate tissue. Maximal expression of both subunits was observed in prostatic carcinomas metastatic to lymph node and bone. Tumor cells that stained positively with anti-α subunit antibodies were also reactive with anti-β subunit antibodies, indicating that they express high-affinity GM-CSF receptors. Our data show that the LNCaP cells express functional GM-CSF receptors and that prostatic carcinomas have prominent GM-CSF receptor expression. These findings imply that both hyperplastic and neoplastic prostatic tissues may be responsive to GM-CSF.