A standard tissue as a control for histochemical and immunohistochemical staining.

The variable quality of histochemical and immunohistochemical staining of tissues may be attributed to pre-analytical and analytical variables. Both categories of variables frequently are undefined or inadequately controlled during specimen collection and preparation. Pre-analytical variables may alter the molecular composition of tissues, which results in variable staining; such variations may cause problems when different tissues are used as staining controls. We developed a standard tissue for use as a staining control. Our standard tissue contains five components: 1) nine combined human cell lines mixed with stroma from human spleen; 2) a squamous cancer cell line, A431; 3) fungus; 4) transverse sections of the mosquitofish and 5) normal human spleen. The first three components were embedded in HistoGel(™) and all components were processed to paraffin and used to construct a single standard paraffin block. The muscles of mosquitofish and arteries of the spleen are positive controls for eosin staining, while other tissues are useful for assessing hematoxylin staining. The mosquitofish tissues also are excellent controls for the Masson trichrome stain and all mucin-related histochemical stains that we tested. The goblet cells of the intestine and skin stained strongly with Alcian blue, pH 2.5 (AB-2.5), mucicarmine, colloidal iron, periodic acid Schiff (PAS) or PAS-hematoxylin (PASH) and combination stains such as colloidal iron-PASH. Cell lines were not useful for evaluating histochemical stains except for PASH. The splenic stroma was a useful control for AB-2.5; however, eosin and mucin stains stained cell lines poorly, probably due to their rapid growth and associated loss of some differentiated characteristics such as production of mucins. Nevertheless, the cell lines were a critical control for immunohistochemical stains. Immunostaining of specific cell lines was consistent with the presence of markers, e.g., EGFr in DU145 cells. The cell lines expressed a wide range of markers, so they were useful controls for immunohistochemical staining including EGFr, HER2, E-cadherin, cytokeratins, Ki67, PCNA, estrogen receptor, progesterone receptor, CD3, CD20 and CD45, activated (cleaved) caspase 3 and Bcl-2. The cell lines also were a control for the TUNEL stain.

Combined effects of formalin fixation and tissue processing on immunorecognition.

It is accepted that aldehyde-based fixation of cells can affect immunodetection of antigens; however, the effects of tissue processing on immunodetection have not been analyzed systematically. We investigated the effects of aldehyde-based fixation and the various cumulative steps of tissue processing on immunohistochemical detection of specific antigens. DU145 (prostate) and SKOV3 (ovarian) cancer cell lines were cultured as monolayers on microscope slides. Immunohistochemical detection of Ki67/MIB-1 and proliferating cell nuclear antigen (PCNA) was evaluated after various fixation times in 10% neutral buffered formalin and after each of the several cumulative steps of tissue processing. The effect of antigen retrieval (AR) was evaluated concomitantly as an additional variable. Our results indicate that in addition to fixation, each of the tissue processing steps has effects on immunorecognition of the epitopes recognized by these antibodies. Extensive dehydration through ethanols to absolute ethanol had only modest effects, except for the detection of Ki67/MIB-1 in SKOV-3 cells where the effect was stronger. In general, however, establishment of a hydrophobic environment by xylene resulted in the greatest decrease in immunorecognition. AR compensated for most, but not all, of the losses in staining following fixation and exposure to xylene; however, AR gave consistent results for most steps of tissue processing, which suggests that AR also should be used for staining PCNA. The cellular variations that were observed indicate that the effects of fixation and other steps of tissue processing may depend on how antigens are packaged by specific cells.

Cell density influences the effect of celecoxib in two carcinoma cell lines.

It has been reported that when ovarian carcinoma cell lines are exposed to various concentrations of celecoxib, a COX-2 inhibitor, cell growth is decreased in a dose dependant manner. To examine further the effect of celecoxib, different cell densities of two carcinoma cell lines were exposed to various concentrations of celecoxib. LNCAP prostate and CAOV3 ovarian carcinoma cells were obtained from the American Type Culture Collection and maintained in Rosewell Park Memorial Institute 1640 and Dulbeceo's modified Eagle's medium, respectively. Each cell line was supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotic-antimycotic solution, and placed in a humidified atmosphere containing 5% CO2 at 37 degrees C. After each cell line reached a confluency of 70-80%, 1,000, 2,000, 3,000, 5,000, 7,000 and 10,000 cells/well were seeded in 96 well plates in 100 microl medium/per well for 24 h. Each cell line was exposed to the same concentrations of celecoxib (10-100 microM) at each cell density for 72 h. Cell growth was assessed using a tetrazolium conversion assay. A significant decrease compared to controls was observed in cell growth at each cell density of LNCAP and CAOV3 cells plated with >or=30 microM and >or=50 microM celecoxib, respectively. When the cell growth curves were compared for each cell density at the same concentration of celecoxib, a significant decrease in cell growth was observed when LNCAP cells were plated at 10,000 cells/well and exposed to 10-100 microM celecoxib. At a cell density >or= 5,000 LNCAP cells/well, the inhibitory effect of celecoxib was less. Similarly, a significant decrease in cell growth was observed in CAOV3 cells plated at 1,000 cells/well compared to other cell numbers plated at the same drug concentrations. At a cell density of > 5,000 CAOV3 cells/well, the inhibitory effect of celecoxib was significantly less compared to other cell densities at the same concentration. We observed a more sensitive decrease in cell growth in both carcinoma lines studied at a cell density of 1,000 cells/well with exposure to 10-100 microM celecoxib. Both carcinoma cell lines were less sensitive at a cell density of 5,000 cells/well. Our results suggest that the inhibitory effect of celecoxib may be affected by cell density. Therefore, careful attention must be paid to determining the appropriate cell density for cytotoxicity studies.

The response of extracellular signal-regulated kinase (ERK) to androgen-induced proliferation in the androgen-sensitive prostate cancer cell line, LNCaP.

The mechanisms by which androgens stimulate proliferation of prostate cancer cells are poorly understood. It has been proposed that androgen stimulation may induce the mitogen-activated protein (MAP) kinase system in prostate cancer cells and lead to cellular proliferation. We attempted to evaluate the role of the extracellular signal-regulated kinase (ERK) pathway in the stimulation by androgens of prostate cancer cell proliferation. Androgen-sensitive prostate cancer cell line (LNCaP) cells plated on sterile glass coverslips were treated with 10(-8) M dihydrotestosterone (DHT) or epidermal growth factor (EGF) (10 ng/ml) for periods ranging from 1 min to 96 h. The proliferative index of the cells, evaluated by immunoperoxidase staining of cells with an antibody to Ki-67, was increased at least two-fold at all time points from 5 min to 48 h following exposure to either DHT or EGF. Immunohistochemical evaluation of ERK1/2 and pERK (activated ERK) demonstrated high levels of ERK1/2 in untreated LNCaP cells, while pERK was expressed at much lower levels. Following treatment with DHT, no change in staining intensity for either ERK1/2 or pERK was observed, while treatment with EGF resulted in no change in ERK1/2, but significantly increased cytoplasmic staining for pERK at all time points beyond 2 min. These results were confirmed by Western blot analysis of ERK1/2 and pERK expression in these cell lines following treatment with DHT or EGF. Our findings suggest that the proliferative response of prostate cancer cells to androgens, unlike the proliferative response to EGF, is not mediated by the activation of ERK1/2, and that currently undefined pathways other than those involving ERK1/2 are involved.

The use of dimethylsulfoxide as a vehicle in cell culture experiments using ovarian carcinoma cell lines.

Dimethylsulfoxide (DMSO) is a well-known solvent that is commonly used in the laboratory. We selected DMSO as the vehicle for an experiment designed to determine if several nonsteroidal anti-inflammatory agents inhibit the growth of Caov-3, OVCAR-3, and SK-OV-3 ovarian carcinoma cell lines. Using the tetrazolium conversion assay, however, we observed some variability in the number of cells present in each ovarian carcinoma cell line with varying concentrations of DMSO (10(-6)-10(-2) M) compared to medium alone. Similarly, when Caov-3, OVCAR-3, and SK-OV-3 cells were treated with 10(-4) M DMSO plus medium (Dulbecco's Modified Eagle Medium with 10% fetal bovine serum) and plated on coverslips, the total number of cells present in 60 random fields increased significantly (P < 0.0001) for each ovarian carcinoma cell line treated with DMSO compared to medium alone. Ethanol did not demonstrate such prominent effects on cellular growth. Our observations are important to consider when selecting an appropriate solvent, especially for growth inhibition studies using Caov-3, OVCAR-3, and SK-OV-3 cell lines.

Altered subcellular localization of suppressin, a novel inhibitor of cell-cycle entry, is an independent prognostic factor in colorectal adenocarcinomas.

PURPOSE: Suppressin (SPN), a novel inhibitor of the entry into the cell cycle, has properties of a tumor suppressor gene; however, its role in the development and progression of a human malignancy is not studied. Therefore, we evaluated the status of spn and its prognostic value in human colorectal adenocarcinoma (CRC). EXPERIMENTAL DESIGN: Inhibition of cell proliferation by exogenous/extracellular SPN was assessed by [(3)H]thymidine incorporation. The genetic status of spn in two colon cancer cell lines (LS180 and WiDr) and in a human CRC was determined using direct cDNA sequencing techniques. Phenotypic expression of SPN was evaluated in 105 CRC archival tissues using immunohistochemical methods. Univariate Kaplan-Meier and multivariate Cox proportional hazards models were used to determine the prognostic significance of SPN expression. RESULTS: Exogenous SPN inhibited the proliferation of the LS180 cell line, which also has a mutation in one allele of the spn gene. The spn gene was also mutated in the primary CRC. Expression of SPN was primarily cytoplasmic in nonmucinous CRCs and nuclear in mucinous CRCs. However, the evaluation of 85 nonmucinous CRCs demonstrated that nuclear localization of SPN, nuclear accumulation of p53, and nodal status were independent prognostic indicators with hazard ratios of 2.34, 2.33, and 3.04, respectively. Nuclear localization of SPN plus nuclear accumulation of p53 formed a stronger prognostic indicator (hazard ratios = 5.45) than local nodal status. CONCLUSIONS: This is the first report of genetic alterations in the spn gene in a human malignancy and suggests that genetic alterations in spn and the resulting immunohistochemical phenotypes based on SPN subcellular localization in CRCs may be useful in determining prognosis of patients with subtypes of CRC.

Altered global methylation of DNA: an epigenetic difference in susceptibility for lung cancer is associated with its progression.

Alterations in global DNA methylation have been observed in many cancers, but whether such alterations represent an epigenetic difference in susceptibility for the disease is unknown. The status of global DNA methylation also has not been reported in intact or specific types of cells involved in the carcinogenic process. To address these issues in lung carcinogenesis, we evaluated the status of global DNA methylation by using a monoclonal antibody specific for 5-methylcytosine (5-mc) in randomly selected lung specimens of 60 cigarette smokers who developed squamous cell carcinoma (SCC) and 30 cigarette smokers who did not. 5-mc immunostaining scores of DNA of SCC (0.61 +/- 0.42) and associated hyperplastic lesions (0.82 +/- 0.27) was significantly lower than those of DNA of histologically normal bronchial epithelial cells (0.99 +/- 0.52) and hyperplastic lesions (1.2 +/- 0.22) of noncancer specimens. The ratio of 5-mc scores between SCC and matched uninvolved bronchial epithelial cells was significantly associated with advanced stage and size of the tumor. The results suggest that alteration in global DNA methylation is an important epigenetic difference in susceptibility for the development of lung cancer. The reduced global DNA methylation in SCC compared with epithelial hyperplasia and its association with tumor size and disease stage is suggestive of its involvement in the progression of SCC. The results also indicate that normal methylation of DNA in epithelial hyperplastic lesions may prevent the transformation of these lesions to invasive cancer. If these results are confirmed, the status of DNA methylation in early lesions such as epithelial hyperplasia could be used to identify smokers who are at risk for the development of SCC.

Fatty acid synthase: an early molecular marker of progression of prostatic adenocarcinoma to androgen independence.

PURPOSE: Changes in fatty acid synthase levels were investigated in the CWR22 xenograft model of prostatic adenocarcinoma after castration and during progression to androgen independence. MATERIALS AND METHODS: Male nude mice with CWR22 tumors were castrated and sacrificed 3, 7, 21, 28 or 42 days after castration. Some mice received testosterone replenishment 21 days after castration and were sacrificed 7 days later. In addition, other castrates were maintained for 7 to 10 months to allow tumors to relapse to androgen independence. Fatty acid synthase was measured by immunohistochemical study and Western blot analysis. RESULTS: Fatty acid synthase decreased rapidly after castration and after 28 to 42 days it was 5% to 10% of the level in tumors of intact controls. Temporal changes in fatty acid synthase after castration were associated with decreased proliferative potential and increased levels of the cyclin dependent kinase inhibitor p27Kip-1. Testosterone treatment of castrates at 21 to 28 days after castration increased fatty acid synthase expression to the level in tumors of intact controls. Tumors of long-term castrates with post-castration androgen independent growth had increased fatty acid synthase, as did small focal areas of androgen independent proliferation in tumors that had not increased in size by 7 to 10 months. In relapsed CWR22 tumors and in focal areas of androgen independent proliferation in nonrelapsed CWR22 tumors increased fatty acid synthase was associated spatially with increased proliferation and decreased p27Kip-1. CONCLUSIONS: Fatty acid synthase in CWR22 tumors depends initially on testosterone and it is associated with androgen mediated proliferation. Furthermore, increased fatty acid synthase levels associated with androgen independent proliferation may represent an early event in the development of the androgen independent phenotype.

Localized folate and vitamin B-12 deficiency in squamous cell lung cancer is associated with global DNA hypomethylation.

We measured the concentrations of folate and vitamin B-12 in paired tissue samples of squamous cell cancer (SCC) and adjacent grossly normal-appearing uninvolved bronchial mucosa (from which SCC developed and also "at risk" of developing SCC) of the lung in 12 subjects to determine the involvement of these vitamins in 1) lung carcinogenesis and 2) global DNA methylation. The folate concentrations were significantly lower in SCCs than in uninvolved tissues (p = 0.03). The vitamin B-12 concentrations were also significantly lower in SCCs than in uninvolved tissues (p = 0.02). The radiolabeled methyl incorporation (inversely related to the degree of in vivo DNA methylation) was significantly higher in SCCs than in uninvolved tissues (p < 0.0001). The correlation between folate and radiolabeled methyl incorporation was inverse and statistically significant in SCCs (p = 0.03). The correlation between vitamin B-12 and radiolabeled methyl incorporation also was inverse and statistically significant in SCCs (p = 0.009). The relationship between tissue vitamin B-12 and DNA methylation was minimal in uninvolved tissues. The relationship between folate and DNA methylation, however, was inverse in uninvolved tissues. In the multiple regression models that included both vitamins, only folate was inversely associated with radiolabeled methyl incorporation in uninvolved and cancerous tissues. These results suggested that folate might be the limiting vitamin for proper DNA methylation in SCC as well as in tissues at risk of developing SCC. Several possible mechanisms of folate deficiency, including inactivation of the vitamin by exposure to carcinogens of cigarette smoke and underexpression or absence of folate receptor in SCCs and associated premalignant lesions, are discussed in light of these findings.

Ep-Cam levels in prostatic adenocarcinoma and prostatic intraepithelial neoplasia.

PURPOSE: The monoclonal antibody (mAb) 323/A3, a second generation high affinity antibody of the 17-1A antibody family, recognizes a 40 kDa transmembrane glycoprotein that has been referred to as Ep-CAM, 17-1A recognized antigen, or EGP40. While Ep-CAM is expressed on the basolateral surface of a variety of epithelia, the strongest expression is frequently detected among several types of carcinoma. In this regard, Ep-CAM may be useful in therapy, in diagnosis, and/or in prognosis. We examined the distribution of Ep-CAM in normal, dysplastic, and malignant prostatic epithelium. MATERIALS AND METHODS: Paraffin sections of prostate tissue from 76 patients with clinically localized (pT2) prostatic adenocarcinoma were immunostained with mouse mAb 323/A3 using the avidin-biotin horseradish peroxidase method. RESULTS: Within benign prostatic epithelium, immunoreactivity typically was low and frequently was restricted to the luminal cells. In contrast, moderate to strong immunostaining was detected frequently in the luminal cells of high grade prostatic intraepithelial neoplasia (PIN). Furthermore, strong immunostaining usually was detected in the cells of adenocarcinomas. The immunostaining in PIN (p<0.0001) and in adenocarcinoma (p<0.0001) was significantly greater than that observed in the normal epithelium. Expression of Ep-CAM did not vary significantly with the Gleason score of tumors or the clinical outcome of patients. Expression of Ep-CAM was demonstrated also in the malignant prostatic cell lines LNCaP, DU145 and PC3 using immunohistochemistry and an immunoblot technique. CONCLUSIONS: These findings suggest that increased levels of Ep-CAM represent an early event in the development of prostatic adenocarcinoma.

Androgenic regulation of growth factor and growth factor receptor expression in the CWR22 model of prostatic adenocarcinoma.

The effects of androgen manipulation on epidermal growth factor (EGF) receptor, p185erbB-2 and transforming growth factor-alpha (TGF-alpha) levels were examined in prostatic adenocarcinoma. Male nude mice were inoculated with the CWR22 androgen-dependent human prostatic tumor or an androgen-independent (CWR22R) derivative. Mice with CWR22 tumors were castrated and subsequently killed at 3, 7, 21, 28 or 42 days post-castration. Other CWR22-bearing mice received s.c. testosterone pellets at 21 days post-castration and were killed 7 days later. EGF receptor, p185erbB-2 and TGF-alpha levels were examined by immuno-histochemistry. Strong EGF receptor and p185erbB-2 immunostaining was detected in CWR22 tumors from intact controls. EGF receptor immunostaining decreased by 65% to 70% at 21 to 42 days post-castration. Testosterone treatment at 21 to 28 days post-castration resulted in a 2-fold increase in EGF receptor immunostaining. p185erbB-2 immunostaining within CWR22 tumors did not decrease following castration and, in fact, was slightly increased at 7 days post-castration. The effects of castration on EGF receptor and p185erbB-2 levels were confirmed by Western blot analysis. Fewer than 10% of CWR22 tumor cells demonstrated strong TGF-alpha immunostaining, and androgen manipulation did not effect TGF-alpha immunostaining. In contrast, 30% of androgen-independent CWR22R tumor cells were strongly immunostained for TGF-alpha. Our findings indicate that EGF receptor levels, but not p185erbB-2 levels, are strongly dependent on testosterone in CWR22 tumors. The co-localization of TGF-alpha and the EGF receptor in CWR22R tumors suggests that these factors may constitute an autocrine pathway that regulates androgen-independent growth.

Changes in cyclin dependent kinase inhibitors p21 and p27 during the castration induced regression of the CWR22 model of prostatic adenocarcinoma.

PURPOSE: The expression of the cyclin dependent kinase inhibitors p21 and p27 was examined in prostatic adenocarcinomas following castration. MATERIALS AND METHODS: Male nude mice inoculated with the androgen dependent human prostatic tumor CWR22 were castrated when the tumors reached a volume of 0.8 to 1.1 cm.3 and were sacrificed at 3, 7, 21, 28 and 42 days post-castration. An additional group of mice received a subcutaneous testosterone pellet at 21 days post-castration and was sacrificed at 28 days post-castration. The expression of the Ki-67 antigen, p21 and p27 was examined by immunohistochemistry. RESULTS: The mitotic rate as well as the number of Ki-67 antigen positive cells decreased to 3% of intact control values by 7 days post-castration and were less than 0.01% of intact control values at 21, 28 and 42 days post-castration. The percentage of p21 expressing cells decreased from 15+/-2% in intact controls to less than 1% by 42 days post-castration. In contrast, the percentage of cells that expressed p27 increased from 25+/-3% in intact controls to 51+/-8% at 3 days post-castration and to 80 to 95% at days 7, 21, 28 and 42 days post-castration. Testosterone treatment from 21 to 28 days post-castration resulted in an increase in Ki-67 antigen positive cells to 200% of intact controls and a concomitant reduction in p27 expressing cells to about 50% of intact controls. Castration-induced changes in p27 expression were not observed in the CWR22R tumor, a transplantable relapsed derivative of the CWR22 tumor. CONCLUSION: These findings suggest that p27 expression is regulated negatively by androgens and that increased expression of p27 in CWR22 xenografts may be involved in the suppression of proliferation following castration.

Hormone levels and mammary epithelial cell proliferation in rats treated with a regimen of estradiol and progesterone that mimics the preventive effect of pregnancy against mammary cancer.

Women who bear their first child by their late teens have about half the risk of developing breast cancer relative to nulliparous women. The rat is a good model for studying the role of hormones in breast cancer since, for example, young rats become nearly refractory to mammary carcinogenesis after delivering a litter of pups. Short term administration of estradiol and progesterone (E & P) provides virgin rats protection from mammary carcinogenesis as effectively as pregnancy. The purpose of these studies were twofold: first, to evaluate potential long-term toxicity of the E & P treatments and second, to compare hormone treated rats and pregnant rats with respect to circulating E & P levels as well as mammary epithelial cell proliferation and differentiation. To test for toxicity, rats were treated with E & P (20 micrograms and 4 mg, respectively) or vehicle by s.c. injections 5 times per week for 5 weeks beginning at 40 days of age. The animals were weighed biweekly and sacrificed at 500 days of age when detailed necropsies were performed. No significant difference in weight gain was observed between the two groups nor was any toxicity grossly observable in the hormone-treated rats. Furthermore, there was no increase in the number of spontaneous mammary or pituitary tumors in the E & P treated group relative to controls. To evaluate serum hormone titers and mammary proliferation, rats were treated with steroids or vehicle daily beginning at 65 days of age. At 6 and 24 hours after the 1st, 14th and 35th injection, serum E & P were measured by RIA and mammary epithelial cell proliferation by immunohistochemistry (PCNA). At 6 hours after each injection, E & P levels were 3 to 5 fold those observed late in pregnancy. By 24 hours, however, E & P levels subsided to late pregnancy levels or lower. The mammary epithelial cell proliferation index in either E & P treated or late pregnant rats was 6 to 14%. Histologic sections and wholemounts of mammary glands showed a similar degree of differentiation between rats treated with E & P for 14 days or longer and late pregnant rats. These data further suggest that E & P treatments are a non-toxic means of mimicking the protective effect of pregnancy against mammary cancer and that pregnancy or hormone treatments may achieve this prophylaxis through a differentiation mechanism.

The effects of dihydrotestosterone on the expression of p185(erbB-2) and c-erbB-2 mRNA in the prostatic cell line LNCaP.

The c-erbB-2 proto-oncogene encodes a 185000 molecular weight protein (p185(erbB-2)) which shares structural homology with the epidermal growth factor (EGF) receptor. We examined the effects of dihydrotestosterone (DHT) on the expression of p185(erbB2) and c-erbB-2 mRNA in the human malignant prostatic cell line LNCaP. LNCaP cells grown in steroid-depleted media were treated with DHT (10(-11)-10(-6) M) for 48 h and p185(erbB-2) expression was determined by Western blotting and immunoprecipitation of 35S-methionine labelled p185(erbB-2). c-erbB-2 mRNA levels were determined using a competitive quantitative reverse transcription-polymerase chain reaction (RT-PCR) based technique. DHT at concentrations of 10(-9) M or greater resulted in decreased expression of p185(erbB-2). In contrast, DHT at these levels stimulated EGF receptor protein expression and cellular proliferation. c-erbB-2 mRNA levels declined to 30-50% of control levels following treatment with DHT of 10(-10) M or greater. Furthermore, the inhibitory effects on c-erbB-2 mRNA were rapid, occurring within 6-12 h of treatment. In summary, these results demonstrate that DHT, at concentrations that stimulate cell growth, inhibits the expression of p185(erbB-2) and c-erbB-2 mRNA.

Elevated serum levels of p105(erbB-2) in patients with advanced-stage prostatic adenocarcinoma.

Expression of a truncated or extracellular form (p105erbB-2) of p185erbB-2 has been demonstrated in the sera of breast cancer patients. We examined the levels of p105erbB-2 in the sera of patients with various stages of prostatic adenocarcinoma, in patients with benign prostatic hyperplasia (BPH) and in a series of control male patients hospitalized for illnesses unrelated to the prostate. p105erbB-2 levels did not differ between the controls and BPH patients or between these groups and patients with stage A, B or C adenocarcinomas. In contrast, serum p105erbB-2 levels of patients with stage D adenocarcinomas were significantly elevated when compared with either control or BPH patients. There was no correlation between PSA and p105erbB-2 levels among controls, patients with BPH or patients with prostate cancer. Patients with poorly differentiated tumors (combined Gleason score >7) or moderately differentiated tumors (combined Gleason score 5-7) had higher p105erbB-2 levels as compared to patients with well-differentiated tumors (combined Gleason score <5), though this difference was not statistically significant. There was no correlation between serum p105erbB-2 levels and p185erbB-2 expression in malignant tissue, as determined by immunohistochemistry. However, patients with moderate to strong expression of p185erbB-2 within the adenocarcinomas were approximately 4 times more likely to demonstrate elevated serum p105erbB-2 levels as compared with patients with low expression of p185erbB-2.

Expression of nm23-H1 in prostatic intraepithelial neoplasia and adenocarcinoma.

The product of the nm23-H1 gene has been reported to be related to the metastatic potential of several tumors. Although several studies have characterized the expression of the nm23-H1 gene product in prostatic adenocarcinoma, little is known of the expression of this protein in prostatic intraepithelial neoplasia (PIN), a putative precancerous lesion. The authors used immunohistochemistry to examine the expression of the nm23-H1 protein in PIN as well as benign and malignant prostatic tissue. A monoclonal antibody to nm23-H1 (Novocastra, Newcastle upon Tyne, UK, clone 37.6) and a biotin/strepavidin detection system were used for antigen localization. Weak to moderate immunostaining was consistently detected in the benign glandular epithelium of 28 radical prostatectomy specimens. In contrast, strong immunostaining was detected in the glandular epithelium in PIN lesions of 19 radical prostatectomy specimens examined. Strong immunostaining was also frequently detected in the malignant cells of 39 localized prostatic adenocarcinomas, as well as 7 metastatic lesions. These findings show a phenotypic similarity of PIN to prostatic adenocarcinoma with respect to nm23-H1 expression. Furthermore, strong expression of nm23-H1 likely represents an early event in the development of prostatic adenocarcinoma.

Accumulation of the p53 protein occurs more frequently in metastatic than in localized prostatic adenocarcinomas.

Mutations of the p53 gene result in increased stability and accumulation of the p53 protein, permitting p53 protein detection by immunohistochemical techniques. We have utilized immunohistochemistry to examine accumulation of the p53 protein at various stages of progression in prostatic adenocarcinomas. p53 protein accumulation was detected using the monoclonal antibody BP53-12-1 in 3 of 28 (11%) localized prostatic adenocarcinomas, and in prostatic intraepithelial neoplasia (PIN) in 1 of 16 (6%) specimens. In contrast, p53 protein was detected in 9 of 16 (56%) primary prostatic adenocarcinomas that were metastatic (stage D), and in 10 of 18 (56%) matching metastases to lymph nodes from these same patients. Thus, we observed a higher incidence of p53 protein accumulation in matching primary and metastatic lesions of patients with stage D adenocarcinoma than in localized (nonmetastatic) adenocarcinomas. We also found that an antigen retrieval solution (ARS) aided in the detection of p53 protein accumulation in prostatic adenocarcinomas. The results indicate that accumulation of p53 protein occurs prior to metastasis, and identifies a subclass of prostatic adenocarcinomas that express a high potential for metastasis.

Expression of p160erbB-3 and p185erbB-2 in prostatic intraepithelial neoplasia and prostatic adenocarcinoma.

BACKGROUND: Although prostatic adenocarcinoma is the most frequent malignancy among American men, little is known concerning the roles of growth factors, growth factor receptors, or proto-oncogene products in the development and progression of this malignancy. PURPOSE: We examined and compared the expression and cellular distribution of the erbB-3 protein (p160erbB-3) and the erbB-2 protein (p185erbB-2) in various stages of development and progression of prostatic adenocarcinoma. METHODS: Immunoperoxidase staining was used to determine the expression of p160erbB-3 and p185erbB-2 in benign prostatic epithelium, prostatic intraepithelial neoplasia, localized adenocarcinomas (pathologic stages B and C) as well as matching primary and lymph node lesions from patients with stage D adenocarcinoma. In order to test antibody specificity, we used Western blot analysis to examine the expression of p160erbB-3 and p185erbB-2 in selected prostatic adenocarcinomas. RESULTS: Within benign glands, immunostaining for p160erbB-3 or p185erbB-2 was strongest in the basal cells and was typically absent or weak in the luminal (secretory) cells. Expression of both proteins within luminal cells was localized to cell membranes. We examined the expression of p160erbB-3 and p185erbB-2 in the prostatic intraepithelial neoplastic lesions of 22 radical prostatectomy specimens. Like benign glands, the basal cells of these neoplastic lesions typically demonstrated strong to moderate immunostaining when stained with either antibody. In contrast to benign glands, moderate to strong immunostaining for p160erbB-3 and p185erbB-2 was frequently observed within the cytoplasm and cell membranes of the prostatic intraepithelial neoplastic luminal cells. p160erbB-3 and p185erbB-2 were detected within the malignant cells in 28 and 29 of 29 localized adenocarcinomas, respectively. Immunostaining with either antibody was typically moderate to strong in the malignant cells. Both proteins were expressed on the cellular membranes as well as in the cytoplasm of malignant cells. Immunostaining for p160erbB-3 was detected in the matching primary and metastatic lesions (lymph nodes) in 10 of 11 patients with stage D adenocarcinoma. Immunostaining for p185erbB-2 was detected in matching primary and metastatic lesions (lymph nodes) obtained from 16 patients with stage D adenocarcinoma. Western blot analysis confirmed the specificity of the antibodies. CONCLUSIONS: These results suggest that increased expression and changes in the subcellular distribution of both p160erbB-3 and p185erbB-2 represent early events in the development and progression of prostatic adenocarcinomas. The high expression and distribution of both p160erbB-3 and p185erbB-2 are retained both in advanced-stage primary and metastatic tumors.