A human melanoma line heterogeneous with respect to metastatic capacity in athymic nude mice.

The ability of the human A375 melanoma cell line to produce experimental and spontaneous in young BALB/c nude mice was examined. Cloned lines, obtained by isolation in semisolid agar, differed significantly (4/10 cloned lines examined, P less than or equal to .005) from the parent tumor line with regard to their ability to form lung tumor nodules subsequent to iv injection. Lines established from such lung tumor nodules were more metastatic than the parent line following both iv and sc injection. These results show that the human melanoma cell line used in these studies contained cells with diverse metastatic potential and suggest that metastasis in the nude mouse results from the preferential selection of metastatic subpopulations.

Metastatic behavior of human tumor cell lines grown in the nude mouse.

The metastatic behavior of seven human tumor cell lines grown in young (3- to 4-week-old) nude mice was studied. Two cell lines were derived from malignant melanomas, one from a colon carcinoma, two from prostate adenocarcinomas, and two from renal adenocarcinomas. Many of the cell lines produced metastases after i.v. injection (experimental metastasis) and after s.c. transplantation (spontaneous metastasis) into young nude mice. The incidence of metastasis seemed dependent primarily on the biological characteristics of the individual tumor cell line. However, the incidence of metastasis of some tumor cell lines could be increased by isolation and establishment of variant sublines from secondary tumor deposits, by prolonged systemic administration of 17 beta-estradiol to suppress natural killer cell activity, and/or by use of an advantageous site of tumor implantation. Intrasplenic injection of tumor cells allowed the most dramatic overall expression of metastatic capacity in these cell lines, resulting in frequent and large metastases to liver, lungs, and the mesenteric, omental, and mediastinal lymph nodes.

Autonomous growth of androgen-independent human prostatic carcinoma cells: role of transforming growth factor alpha.

The androgen-independent prostatic carcinoma cell line PC3 is known to exhibit autonomous growth in vitro and in vivo. The purpose of the present study was to investigate the role of transforming growth factor alpha (TGF-alpha) and its receptor, the epidermal growth factor (EGF) receptor, in the regulation of PC3 cell proliferation. Results showed that PC3 cells secrete factors into conditioned medium that are mitogenic for the less aggressive prostatic carcinoma lines DU145 and LNCaP. Gel filtration chromatography of PC3-conditioned medium revealed a major peak of mitogenic activity at a molecular weight of 5,000 to 10,000 which was inhibited by the addition of antibody to TGF-alpha. The synthesis and secretion of TGF-alpha by PC3 cells were further demonstrated by immunoblotting and radioimmunoassay. Radioreceptor analysis showed a single class (Kd 5.3 nM) of EGF receptors on PC3 cells. The presence of Mr 170,000 EGF receptors on PC3 cells was further demonstrated by immunoprecipitation of metabolically labeled proteins. TGF-alpha was effective in stimulating the growth of low-density, but not high-density, PC3 cultures. In addition, the proliferation of PC3 cells under serum-free defined conditions was inhibited by antibodies to TGF-alpha and/or the EGF receptor. These data indicate that TGF-alpha/EGF receptor interactions are partially responsible for autonomous growth of the PC3 cell line and may explain one mechanism of escape from androgen-dependent growth in human prostatic carcinoma.

Epidermal growth factor receptor monoclonal antibody inhibits constitutive receptor phosphorylation, reduces autonomous growth, and sensitizes androgen-independent prostatic carcinoma cells to tumor necrosis factor alpha.

Results of recent studies indicate that cultured, androgen-independent prostatic carcinoma cells synthesize and secrete transforming growth factor alpha, which interacts with epidermal growth factor receptors (EGFRs) to promote autonomous growth. In the present study, we evaluated the expression and constitutive activation of EGFRs in normal prostatic epithelial cells and the androgen-independent prostatic carcinoma cell lines PC3 and DU145. Our studies showed that cultured normal epithelial cells and androgen-independent prostatic carcinoma cells actively synthesize and exhibit constitutive phosphorylation of the M(r) 170,000 EGFR. The addition of monoclonal anti-EGFR reduced receptor phosphorylation and significantly inhibited the proliferation of prostatic tumor cells. The observed reduction in EGFR phosphorylation could be partially attributed to an antibody-induced decrease in the expression of metabolically labeled EGFR. Results of further studies showed that anti-EGFR enhanced the sensitivity of PC3 cells to the cytotoxic and cytostatic effects of tumor necrosis factor alpha. These studies demonstrate that constitutive activation of EGFR in androgen-independent prostatic carcinoma plays a functional role in the regulation of cellular proliferation in vitro. In addition, the enhanced sensitivity of prostatic carcinoma cells to tumor necrosis factor alpha in the presence of anti-EGFR provides a rationale for the further investigation of combination therapy in the treatment of disseminated, androgen-independent disease.

A rapid in vitro assay for quantitating the invasive potential of tumor cells.

We have reconstituted a matrix of basement membrane onto a filter in a Boyden chamber and assessed the ability of various malignant and nonmalignant cells to penetrate through the coated filter. Cells from all the malignant cell lines tested were able to cross the matrix in 5-6 h, whereas human fibroblasts as well as mouse 3T3 and 10T1/2 cell lines, which are not tumorigenic, were not invasive. In addition, normal primary prostate epithelial cells and benign prostatic hyperplasia cells were not invasive when tested in this assay, whereas malignant prostate carcinoma cells were highly invasive. Parallel experiments with these prostatic cells using the intrasplenic assay for metastasis detection in the nude mouse confirmed the benign behavior of the former cells and the metastatic phenotype of the latter ones. These results suggest that this in vitro test allows the rapid and quantitative assessment of invasiveness and a means to screen for drugs which alter the invasive phenotype of tumor cells.

Prevention of cell death induced by tumor necrosis factor alpha in LNCaP cells by overexpression of sulfated glycoprotein-2 (clusterin).

Sulfated glycoprotein-2 (SGP-2) expression has been associated with programmed cell death in the prostate, but its exact role remains unclear. The present study was carried out in an attempt to establish the function of SGP-2 in programmed cell death using tumor necrosis factor (TNF) alpha-induced cytotoxicity in LNCaP cells as the model system. LNCaP is an androgen-sensitive, human prostatic cancer cell line that responds to TNF in culture by undergoing programmed cell death, as determined by the loss of cell number, failure to exclude trypan blue, detection of DNA fragmentation, and increased release of previously incorporated [3H]thymidine. Immunocytochemical staining for SGP-2 was weak but evident in LNCaP cells. Following treatment with TNF alpha, there was a time-dependent increase in SGP-2 staining, the intensity of which peaked at 2 h and declined thereafter. SGP-2 staining in LNCaP cells was undetectable prior to the onset of DNA fragmentation at 6 h of TNF treatment. This observation indicated that TNF-induced cell death in LNCaP cells was characterized by an initial transient elevation of SGP-2, followed by a period of SGP-2 depletion that preceded cell death. Transfection of LNCaP with a 21-base oligonucleotide antisense to SGP-2 resulted in a significant increase in cell death that was sequence specific and was accompanied by a reduction in SGP-2 biosynthesis. These findings supported the concept that SGP-2 depletion, rather than its expression, was associated with cell death. Finally, stable transfection and subsequent overexpression of SGP-2 in LNCaP cells resulted in resistance to the cytotoxic effect of TNF. These results have provided evidence to indicate that SGP-2 plays a role in the protection of TNF-induced cell death in LNCaP cells.

Loss of expression of transforming growth factor-beta receptors is associated with poor prognosis in prostate cancer patients.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of proliferation in most cells and exerts its effects through an interaction with membrane receptors type I (TGF-betaRI) and type II (TGF-betaRII). Recently, we have demonstrated a correlation between the loss of expression of TGF-betaRI and TGF-betaRII and increasing Gleason score in archival human prostate cancer tissues. To evaluate the potential prognostic value of this observation, the present study investigated the expression of TGF-beta receptors in association with disease progression after the initial diagnosis in 52 archival human prostate cancer tissues. The expression of both TGF-betaRI and TGF-betaRII was correlated with the Gleason score, clinical tumor stage, 4-year survival rate, and serological recurrence rate after radical prostatectomy. Results revealed that there was a significant association between the Gleason score and the loss of expression of TGF-betaRI (P < 0.025) and TGF-betaRII (P < 0.01). However, only the loss of TGF-betaRI expression showed a statistically significant association with the clinical tumor stage (P < 0.05), 4-year survival rate (P < 0.05), and serological recurrence rate after radical prostatectomy (P < 0.025). Therefore, these data indicate that the loss of TGF-betaRI expression as measured by immunohistochemical staining may be a potential prognostic marker in prostate cancer patients.

Intracellular levels of SGP-2 (Clusterin) correlate with tumor grade in prostate cancer.

Our previous observations in LNCaP cells in vitro demonstrated an association between apoptotic cell death resistance and SGP-2 (Clusterin) overexpression. Accordingly, we hypothesized that high levels of cellular SGP-2 would aid in identifying biologically aggressive prostate cancer cells with unique survival advantages. To test this hypothesis, 40 archival radical prostatectomy and/or biopsy specimens of varying grades of prostate cancer were subjected to immunohistochemical SGP-2 staining. The resulting epithelial stains were quantified subjectively on a scale of 1-3 by four independent observers. Benign prostatic epithelial cells from young donors served as controls and showed a consistently weak staining intensity. In contrast, prostate cancer specimens showed varying degrees of staining intensity that correlated with a Gleason pattern (P = 0.006). This correlation supports the hypothesis that protection from apoptotic death may account, in part, for biologically aggressive tumor behavior.

Loss of expression of transforming growth factor beta type I and type II receptors correlates with tumor grade in human prostate cancer tissues.

Transforming growth factor beta1 (TGF-beta1) is a potential regulator of prostate cancer cell growth that signals through a heteromeric complex composed of type I and type II receptors. In the present study, an attempt was made to establish a correlation between expression of TGF-beta receptors and tumor grade in archival human prostate cancer tissues. To this end, immunohistochemical studies for TGF-beta receptors were carried out on 32 cases of human prostate cancer and 8 samples of benign human prostate. In both benign and malignant human prostate tissues, immunoreactivity for both type I and type II receptors was detected predominantly in epithelial cells. In addition, there was an inverse correlation between the loss of expression of TGF-beta1 type I and type II receptors and the tumor grade. Of the 32 prostate cancer cases screened, staining was completely absent in four samples for type II receptor (P < 0.05) and eight samples for type I receptor (P < 0.025). In contrast, all eight samples of benign prostate tissues investigated in this study showed strong staining for both type I and type II receptors. These results, taken together, indicate that human prostate cancer cells frequently have loss of expression of TGF-beta type I and/or type II receptors. Furthermore, these observations provide a potential mechanism for prostate cancer cells to escape the growth-inhibitory effect of TGF-beta.

Basic fibroblast growth factor: a potential mediator of stromal growth in the human prostate.

Studies were undertaken, using isolated prostatic epithelial and stromal cells, to evaluate the role of basic fibroblast growth factor (bFGF) in the regulation of benign prostatic growth. bFGF was detected in lysates, but not the conditioned media, of cultured prostatic epithelial and stromal cells by Western immunoblotting and immunoprecipitation of metabolically labeled proteins. Immunofluorescence analysis of benign human prostate localized the majority of bFGF to the prostatic stroma. In addition, bFGF was a potent stimulator of stromal cell proliferation in vitro, but was not mitogenic to cultured epithelial cells. Further studies demonstrated bFGF receptors (Kd = 258 pM; 61,400 receptors/cell) on stromal cells, but not epithelial cells. Epithelial cell-conditioned medium was mitogenic for stromal cells, suggesting the presence of paracrine interactions. However, bFGF does not appear to be the mediator of this interaction, since the mitogenic effect of epithelial cell-conditioned medium on stromal cells was not significantly reduced by the addition of anti-bFGF. Additional studies showed that concentrated stromal cell-conditioned medium was not mitogenic to cultured stromal cells under serum-free defined conditions, indicating the lack of an external autocine mechanism. These studies demonstrate that bFGF is actively synthesized by isolated prostatic epithelial and stromal cells, but is largely not secreted. Prostatic stroma, but not epithelia, are responsive to the mitogenic effect of bFGF in vitro. However, because of the limited secretion of bFGF by prostatic cells, the mechanism(s) of bFGF-mediated regulation of stromal growth remains unclear.

Regulation of proliferation and production of prostate-specific antigen in androgen-sensitive prostatic cancer cells, LNCaP, by dihydrotestosterone.

LNCaP is an androgen-sensitive human prostatic cancer cell line. The effect of androgen on these cells is characterized by a bell-shaped growth response and a dose-dependent induction of prostate-specific antigen (PSA) production. The present study was carried out to gain further insight into the effect of androgen on LNCaP. Cells were cultured in phenol red-free RPMI-1640 supplemented with 10% charcoal-stripped fetal bovine serum, with concentrations of dihydrotestosterone (DHT) ranging from 0-10(-7) M, in a 4-day culture system. A bell-shaped growth response was reproduced with a peak level of cell count at 10(-10) M DHT. PSA secretion from these cells did not increase significantly until the DHT level in the medium reached 10(-9) M. A progressive increase in PSA secretion was observed at higher DHT concentrations accompanied with a progressive decline in cellular proliferation. The results of immunocytochemical analysis of PSA localization indicated that the proportion of cells with positive staining for PSA also increased with increasing concentrations of DHT. Analysis of androgen receptors, as determined by both immunocytochemistry and Western blot analysis, showed a decline in nuclear androgen receptor at low concentrations of DHT and an increase in the amount of receptor protein at high concentrations. These results indicated that the androgen-induced bell-shaped growth response in LNCaP cells represented the manifestation of two different cellular events in dose-related manner: cellular proliferation at low DHT concentrations and increased production of PSA at high DHT concentrations.

Negative-lens field expander for patients with concentric field constriction.

An oversized, negative-power spectacle lens may be used as a field expander to improve mobility and general orientation ability of patients with peripheral field loss but intact central vision. The device is inexpensive and simple to use for brief spotting periods.

A simple, inexpensive method to improve low visual acuity of highly hypermetropic, aphakic patients.

An inexpensive optical device, an eyeglass loupe readily available commercially, can be used to improve the near and distance visual acuities of highly hypermetropic, aphakic patients. The device is simple to use and can replace the expensive, conspicuous telescopic system (Bioptic) of similar overall power.

Obturator hernia: an elusive diagnosis.

Obturator hernia is an uncommon problem that occurs predominantly in elderly, debilitated women. The diagnosis should be suspected in such patients who have evidence of small bowel obstruction, often initially intermittent, and who complain of pain along the anteromedial aspect of the thigh. We present experience with three patients that characterizes the difficulties in diagnosis and treatment. Most patients require resection of the incarcerated bowel. Mortality is variable and is related to the condition of the patient.

DNA profile of nephrogenic adenoma assessed by flow cytometry.

Nephrogenic adenoma is a metaplastic condition which usually involves the bladder urothelium within the clinical setting of chronic inflammation. Its presentation/endoscopic appearance may mimic bladder cancer and a premalignant potential has been speculated. DNA flow cytometry performed on involved bladder tissue revealed a diploid phenotype exhibiting low proliferative activity. These findings are most suggestive of a benign process.

Management of distant solitary recurrence in the patient with renal cancer. Contralateral kidney and other sites.

About 25% to 57% of patients with RCC exhibit overt evidence of metastatic disease at the time of initial presentation. These patients have an average survival of about 4 months, and only 10% of them survive 1 year. Metachronous metastatic disease may develop in approximately 50% of patients who have undergone a presumably curative radical nephrectomy. Seventy percent of these patients relapse within the first year and manifest a median survival of about 11 months. The incidence of solitary metastatic lesions in patients with RCC ranges from 1.6% to 3.6%. In reality, the vast majority of these patients have evidence of subclinical, multifocal, micrometastatic disease and ultimately succumb to metastatic RCC. However, a 5-year survival rate of 35% to 50% can be achieved in properly selected patients using aggressive surgical treatment as a major component of therapy. In large part, insightful patient selection is predicated upon an understanding of the unique biologic and clinical issues relevant to each organ site of involvement.

Etiology of benign prostatic hyperplasia.

Benign Prostatic Hyperplasia (BPH) is the most common neoplastic condition that afflicts men, and it constitutes a major factor impacting the health of the American male. This article reviews voiding dysfunction and the role of aging, the testis, and androgen in the development of BPH. Emphasis is placed on new concepts in the basic aspects of BPH etiology as a result of recent investigations.

Advanced prostatic carcinoma. Early versus late endocrine therapy.

Since the landmark observations of Huggins and Hodges in 1941, androgen deprivation has been the mainstay of treatment for advanced-stage prostate cancer. Although early, poorly controlled studies suggested enhanced survival with hormonal therapy, this view fell into disfavor as a result of the observations of the first and second VACURG studies. Recently, there has been a proliferation of experimental and clinical data supporting early androgen deprivation, including a reanalysis of the VACURG data, which suggests a survival advantage for younger patients with stage D disease and high-grade tumors who undergo androgen-ablative therapy at the time of diagnosis. The risk-benefit analysis presented in this review is strongly supportive of early hormonal therapy. Finally, long-term survival of patients with metastatic prostate cancer will require the development of novel treatment strategies effective against androgen-resistant tumor cells and their use in concert with early androgen deprivation.

Effect of spermatocele fluid on growth of human prostatic cells in culture.

The present study was carried out to investigate whether testicular fluid derived from a spermatocele contains substance(s) that promote the growth of human prostatic cells in culture. Human spermatocele fluid was centrifuged to sediment spermatozoa. The supernatant was then added to cultures of human prostatic stromal or epithelial cells that were isolated from surgical specimens of benign prostatic hyperplasia. Addition of spermatocele fluid in quantities of 1 microgram/ml of protein resulted in a significant increase in the number of both prostatic stromal and epithelial cells at the end of a 6-day culture period. Human serum at equivalent protein concentrations in the culture medium had no stimulatory effect. At least two separate growth-promoting factors were found in spermatocele fluid, one for stromal cells and one for epithelial cells. The mitogen for stromal cells was heat labile and persisted after treatment with activated charcoal. The factor for epithelial cells was heat stable but was removed by charcoal treatment. These observations are consistent with the concept that the human testis secretes nonandrogenic substances that can promote prostatic growth.

Clusterin as a possible predictor for biochemical recurrence of prostate cancer following radical prostatectomy with intermediate Gleason scores: a preliminary report.

Disease recurrence following radical prostatectomy is a major concern in prostate cancer patients. Gleason scores are useful in predicting recurrence. Low Gleason scores are usually associated with long disease-free intervals, while high Gleason scores are suggestive of early recurrence. However, prediction of recurrence has been difficult with intermediate Gleason scores. Clusterin is a ubiquitous secretory sulfated glycoprotein. It is also an antiapoptotic mediator in prostate cancer. The objective of the present study is to determine if clusterin can serve as a predictive biomarker for recurrence of prostate cancer with intermediate Gleason scores in patients following radical prostatectomy. Prostatic specimens with Gleason score of 6 (3+3) or 7 (3+4) were obtained from the archival bank. Three groups of specimens were investigated. The first group was from nine patients who developed recurrent disease according to a persistent rise of serum PSA within 3 years following radical prostatectomy. Those in the second group and the third group were from patients who showed no evidence of disease recurrence for at least 5 y (11 patients) and 10 y (eight patients), respectively following the surgery. Histological sections were subjected to immunohistochemical staining using a monoclonal antibody specific for clusterin. The staining intensity was scored as 0, 1, 2, and 3, with 0 being no staining, 1 showing less than 25% positive staining, 2 being 25-50% positive, and 3 showing greater than 75% positive staining. One-way ANOVA with Bonferroni correction was used for statistical analysis. Evaluation of the scores of clusterin staining was carried out according to four specific areas in each specimen. They were (a) benign epithelial cells, (b) malignant epithelial cells (cancer epithelia), (c) stromal cells surrounding benign cells, and (d) stromal cells surrounding malignant cells (cancer stroma). Staining score in prostatic epithelial cells, benign as well as malignant, showed no significant relationship among the three patient groups. However, when staining scores in stromal cells were compared, there was a significant difference between patients with recurrent disease and those showed no evidence of disease recurrence for at least 10 y. Results of this preliminary study support the important role of clusterin in the stromal component for prostate cancer progression. Clusterin immunostaining may be useful to aid the prediction of chance of disease recurrence in patients with Gleason score 6 or 7 prostate cancer following radical prostatectomy. Further studies with a large number of cases are warranted to verify this preliminary finding.