CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation.

Using inverse polymerase chain reaction, we identified CD44, located on chromosome 11p13, as a novel translocation partner of IGH in 9 of 114 cases of gastric, nongastric extranodal, follicular, and nodal diffuse large B-cell lymphoma (DLBCL). Notably, these translocations involving IGHSmu were detected in follicular lymphomas and exclusively in germinal center B cell-ike (GCB)-DLBCLs. CD44 is not expressed in reactive GC B cells. The IGHSmu/CD44 translocations substitute Smu for the CD44 promoter and remove exon 1 of CD44, resulting in the overexpression of Imu-CD44 hybrid mRNA transcripts activated from derivative 11 that encode a new CD44 variant lacking the leader peptide and with a unique C-terminus (CD44DeltaEx1). When overexpressed in vitro in the CD44(-) GCB-DLBCL cell line BJAB, CD44DeltaEx1-green fluorescent protein localized to the cytoplasm and nucleus, whereas CD44s-green fluorescent protein (standard form) localized to the plasma membrane. The ectopic expression of CD44DeltaEx1 in BJAB cells enhanced their proliferation rate and clonogenic ability, indicating a possible pathogenic role of the translocation.

Colchicine and 2-methoxyestradiol Inhibit Human Angiogenesis.

BACKGROUND: Angiogenesis is a critical determinant of tumor growth and the development of metastases. Tubulin inhibitors have been shown to be effective inhibitors of angiogenesis. We hypothesized that colchicine, a well-know tubulin inhibitor and 2-methoxyestradiol (2 MeOH), a novel tubulin inhibitor, would limit the initiation of a human angiogenic response and would limit subsequent neovessel growth in a dose-dependent manner. METHODS: To test this hypothesis, we cultured full-thickness human placental vein discs from three placentas in a fibrin-thrombin clot model. Both colchicine and 2 MeOH were tested over a wide range of concentrations (10(-6) to 10(-12) M) to determine their effect on the percent of wells that initiated an angiogenic response (%I) and the subsequent growth (Angiogenic Index, 0-16 range) of vein-derived neovessels. RESULTS: Colchicine at doses of 10(-6) and 10(-8) M completely inhibited the angiogenic response (CI: 95%, P < 0.0001) but lower (10(-10) to 10(-12) M) doses did not significantly inhibit angiogenesis (P = NS). Effective in vitro colchicine levels far exceed achievable non-toxic human plasma levels. In contrast, 2-methoxyestradiol decreased initiation and angiogenic growth significantly at 10(-6) M (CI: 95%, P < 0.0001), but did not significantly decrease angiogenesis at doses of 10(-8), 10(-10), or 10(-12) M. In contrast to colchicine, human plasma levels of 10(-6) M 2 MeOH are achievable clinically with little or no associated toxicity. CONCLUSIONS: Effective in vitro drug levels of 2 MeOH can be achieved in vivo, suggesting that 2 MeOH may have a role in the clinical treatment of angiogenesis-dependent diseases.

Frequent deletion of Fas gene sequences encoding death and transmembrane domains in nasal natural killer/T-cell lymphoma.

Nasal natural killer (NK)/T-cell lymphoma (NL) frequently co-expresses Fas (Apo-1/CD95) and Fas ligand (FasL), but the tumor cells seldom undergo apoptosis. To determine the reason for failure of apoptosis, we examined Fas mRNA expression in 23 NL cases by reverse transcriptase-polymerase chain reaction and sequenced the entire coding region of the Fas gene in 15 of these cases for which the full-length Fas cDNA could be amplified. The reverse transcriptase-polymerase chain reaction analysis revealed that all of the 23 cases expressed Fas mRNA and the sequencing results showed that in addition to the commonly expressed wild-type Fas mRNA and four alternative splice variants detected in 7 cases, mutant Fas transcripts were present in 9 of the 15 (60%) cases sequenced. With confirmation of some Fas mutations at the gene level, 12 deletions in nine cases and one insertion in one case were eventually identified. To rule out any potential polymerase chain reaction artifacts, the same protocol was used to examine 10 reactive tonsils as a control. No aberrant transcripts associated with deletions were detected in these tonsils except for three alternative splice variants. All of the deletion variants detected in NL contained N-terminal preligand assembly domain but not C-terminal death domain and/or transmembrane domain. Co-detection of the wild-type allele and the mutated Fas alleles without the death domain suggested that a dominant-negative mechanism could block the apoptosis signaling. Moreover, loss of the transmembrane domain could protect the tumor cells from apo-ptosis by producing a soluble form of the Fas receptor. The actuarial 3-year survivals leveled off at 15% for patients carrying the Fas mutations and/or splice variants in the lesions and 49% for those carrying the wild type only, but the difference did not reach statistical significance on the univariate analysis (P = 0.396). Taken together, the findings in this study suggest that frequent Fas gene mutations in NL can result in resistance to apoptosis and may contribute to the pathogenesis of NL by adding to the tumor immune privilege.

Compassion and patient centred care.

BACKGROUND: The essence of medicine is a relationship based upon a concern for suffering. Western medicine, arising from a modernistic philosophy, has a tradition of paternalistic 'doctor centred' care. There are significant criticisms of this approach. OBJECTIVE: Drawing on postmodern perspectives, this article discusses the nature of patient centred care. Patient centred care requires a reconciliation of the patient's and doctor's agenda via attention to communication, power and patient autonomy. Patient centred care has been defined by six domains: the illness experience, the context, finding common ground, partnership, health promotion, and consultation limitations. DISCUSSION: Patients strongly desire patient centred care. It has been associated with improved patient and doctor satisfaction, greater compliance, fewer investigations, referrals and malpractice complaints, and no change in consultation time. Patient centred care exerts a positive influence on health outcomes and is especially applicable in general practice, providing an efficacious and compassionate response to suffering.

Antiangiogenic therapy with somatostatin receptor-mediated in situ radiation.

Tumor growth and the development of metastases require an angiogenic response. Angiogenic vessels uniquely express somatostatin subtype 2 (sst 2) receptors that can transport somatostatin or its analogs into the cell. We hypothesized that radiolabeled somatostatin analogs could inhibit the angiogenic response by selectively destroying proliferating endothelial cells. We evaluated the antiangiogenic effects of 111In-pentetreotide, an sst 2-preferring somatostatin analog in a human vessel model. Disks of human placental vein were embedded in fibrin gels in culture and observed for angiogenic sprouting for 14 days. Vein disks were treated with 111In-pentetreotide (1.5, 15, and 150 microCi/mL) on the day of implantation. Control groups included disks treated with nutrient medium alone, with 111In-chloride, and with unlabeled pentetreotide. The percentage of wells that initiated an angiogenic response and the overall length and density of neovessel sprouts were assessed on Day 14. 111In-pentetreotide treatment did not completely block initiation of the angiogenic response but significantly decreased the growth of neovessels after initiation. Both the receptor-specific Auger electron-induced and nonspecific gamma radiation-mediated effects contributed to the angiotoxicity.

Antitumor and antiangiogenic effects of somatostatin receptor-targeted in situ radiation with (111)In-DTPA-JIC 2DL.

INTRODUCTION: Expression of somatostatin receptor subtype 2 (sst 2) in angiogenic tumor vessels appears to be homogeneous, while tumor cell expression of this receptor is often heterogeneous. We have developed a novel in vitro three-dimensional tumor angiogenesis model to study the antitumor and the antiangiogenic effects of radiolabeled somatostatin analogs. We hypothesized that targeted in situ radiation with an Auger electron-emitting radiolabeled somatostatin analog would produce receptor-specific cytotoxicity in sst 2-expressing cells. MATERIALS AND METHODS: IMR-32 human neuroblastoma (sst 2-positive) and MDA MB-231 human breast cancer (sst 2-negative) xenografts were created in nude mice from monolayer cell cultures. Fragments of these tumors were embedded in three-dimensional fibrin gels supplemented with endothelial growth media and incubated for a period of 14 days. Tumor fragments were treated with 50 microCi/ml of (111)In-JIC 2DL, a sst 2-preferring somatostatin analog, or medium on Day 1. Initial angiogenic activity was determined at 48 h and the mean angiogenic score and tumoricidal responses were assessed on Day 14. RESULTS AND CONCLUSION: Tumoricidal effects of (111)In-JIC 2DL were seen only in sst 2-positive IMR-32 tumors. However, the angiogenic response was inhibited in both IMR-32 and MDA MB-231 tumors independent of the tumor cells' sst 2 status. Somatostatin receptor-mediated in situ radiation therapy has profound cytotoxic effects on angiogenic blood vessels and sst 2-expressing tumor cells.

Inhibition of human angiogenesis with heparin and hydrocortisone.

Angiogenesis is a critical determinant of tumor growth and the development of metastases. Heparin, steroids, and heparin/steroid combinations have been used in a variety of in vitro models and in vivo in animal models as effective inhibitors of angiogenesis. We tested heparin, steroid and heparin/steroid combinations at a variety of concentrations to determine their effect on the human 'angiogenic switch' from a resting to a proliferative endothelium in vessels from three placentas (initiation), and the effect of these compounds on the subsequent growth of a human angiogenic response (promotion). Using full-thickness human placental vein discs cultured in three-dimensional fibrin-thrombin clots, we demonstrated that heparin (300, 3000 micrograms/ml), steroid (350, 3500 micrograms/ml), and combinations of heparin/steroid at these doses effectively blocked both initiation and promotion of a human angiogenic response in a dose-dependent fashion. We also demonstrated that high-dose steroid or heparin/steroid treatment for 15 days resulted in disruption of vessel integrity, while treatment with heparin alone produced a suppressed growth rate but had intact vessel architecture. High-dose heparin/steroid treatment could also disrupt a developed angiogenic response and retard further development of an angiogenic response following the cessation of treatment.

Progressive nuclear translocation of somatostatin analogs.

UNLABELLED: Optimal cancer radiotherapy using Auger electron emitters requires selective localization of radionuclides in close proximity to tumor DNA. METHODS: Intracellular trafficking of (125)I-Tyr1-somatostatin-14 somatotropin-release inhibiting factor (SRIF) and 2 of its analogs, (125)I-WOC 4a and (111)In-pentetreotide, was studied in human neuroblastoma cells. RESULTS: After 24-h incubation, SRIF was degraded or recycled, whereas its protease-resistant analogs progressively accumulated in nuclear fractions. (111)In-pentetreotide binding to DNA increased over time in somatostatin receptor-positive cells but not in somatostatin receptor-negative cells. CONCLUSION: These in vitro studies show that prolonged exposure to radiolabeled SRIF analogs significantly increases their cellular internalization, nuclear translocation, and DNA binding. Clinically, infusion of radiolabeled somatostatin analogs may enhance tumor uptake and retention and provide more effective in situ radiotherapy.

Multiply radioiodinated somatostatin analogs induce receptor-specific cytotoxicity.

BACKGROUND: Radiolabeled somatostatin analogs have gained popularity for tumor imaging and have recently been used for the treatment of somatostatin receptor-expressing tumors. We have developed a novel, N-terminally extended, multiply iodinated somatostatin analog, 125I-WOC 4a, that we hypothesize will be a useful tool for the detection of and therapy for somatostatin receptor-positive tumors. To evaluate the therapeutic potential of this agent, we compared the cytotoxicity of 125I-WOC 4a in a somatostatin receptor subtype-2 (sst 2)-expressing human neurobalstoma cell line to its cytotoxicity in a somatostatin receptor-negative human pancreatic carcinoma cell line. METHODS: IMR-32 neuroblastoma cells (sst 2-positive) and PANC-1 human pancreatic cells (sst 2-negative) were incubated with 125I-WOC 4a at doses ranging from 0.1-100 CPM/cell for 48 h and cell viability was assessed by a colorimetric (MTT) cell viability assay. Subsequently, IMR-32 cells were incubated with either control medium, 125I-WOC 4a (1 cpm/cell) alone, 125I-WOC 4a with 10(-6) M octreotide acetate, 125I (1 cpm/cell) alone, 125I with octreotide acetate, or octreotide acetate alone for 48 h, washed, and cryopreserved for 4 weeks. Cells were then thawed, replated, and allowed to acclimate for 48 h. Cell viability was assessed by trypan blue exclusion and a colorimetric assay. RESULTS: Following short-term exposure, 125I-WOC 4a induced dose-dependent cytotoxicity in IMR-32 cells (P < 0.05 by ANOVA), but not in the PANC-1 cells. After exposure to 125I-WOC 4a (1 cpm/cell) for 48 h followed by a 4-week cryopreserved exposure, significant cytotoxicity was induced in IMR-32 cells (P < 0.05 by ANOVA) which was not seen in cells treated with 125I alone or 125I with 10(-6) M octreotide acetate. Simultaneous exposure to 125I-WOC 4a and octreotide acetate was also cytotoxic. CONCLUSION: 125I-WOC 4a induces receptor-specific cytotoxicity following both short- and long-term drug exposures. This radiopharmaceutical may be useful for localizing or treating somatostatin receptor-positive tumors.

Antisense c-myc retroviral vector suppresses established human prostate cancer.

Prostate cancer eventually becomes androgen resistant, resumes growth, and kills the patient. Characterization of genetic events that lead to androgen refractory prostatic neoplasia has revealed the frequent overexpression of c-myc and uncontrolled prostate cancer proliferation. A novel strategy to combat advanced prostate cancer utilized a replication incompetent retrovirus that contained the mouse mammary tumor virus (MMTV) promoter within the retroviral vector to allow transcription of antisense c-myc gene within target prostate tumor cells. The transduction of cultured DU145 cells by XM6:MMTV-antisense c-myc RNA retrovirus did not affect cell proliferation in culture, yet a single direct injection of MMTV-antisense c-myc viral media into established DU145 tumors in nude mice produced a 94.5% reduction in tumor size compared to tumors treated with control virus MTMV sense fos and untreated tumor by 70 days. Two animals in the antisense c-myc-treated group had complete regression of their tumors. Histopathological examination of the tumors revealed that MMTV-antisense c-myc-transduced DU145 tumors had increased tumor cell differentiation, decreased invasion, and a marked stromal response. The mechanism for the antitumor effect of MMTV-antisense c-myc retrovirus appears to be suppression of c-myc mRNA and protein, and decreased bcl-2 protein. The in vivo transduction of prostate cancer cells with MMTV-antisense c-myc retroviruses reduced tumor growth by suppressing c-myc, resulting in the down-regulation of bcl-2 protein. Consequently, the MMTV-antisense c-myc retrovirus may be useful for gene therapy against advanced, hormone-refractory prostate cancer.

Immunohistochemical localization of TGF beta 1, TGF beta 2, and TGF beta 3 in normal and malignant human prostate.

BACKGROUND: Prostate cancer eventually becomes androgen-independent, suggesting that growth factors such as TGF beta 1-3 may potentially contribute to prostate neoplasia. The pattern and level of TGF beta 1-3 protein expression in normal and malignant human prostate are unknown. METHODS: An immunohistochemical study was undertaken to analyze TGF beta 1, TGF beta 2, and TGF beta 3 protein in malignant and adjacent normal prostates from 25 patients who had clinically localized prostate cancer. RESULTS: Normal prostate exhibited similar TGF beta 1 immunostaining in stromal and epithelial cells, whereas TGF beta 2 and TGF beta 3 protein staining was greater in the epithelial relative to the stromal compartments. In malignancy, prostate epithelial cells had higher TGF beta 1 and TGF beta 2 immunostaining than either the surrounding stromal cells or their normal prostatic epithelial counterparts. Although TGF beta 3 staining intensity was similar for both malignant and normal prostate epithelial cells, the pattern of staining switched from uniform apical to diffuse protein staining in malignant prostate glands. CONCLUSIONS: Prostate cancer was associated with alterations of TGF beta 1, TGF beta 2, and TGF beta 3 expression by prostatic epithelial cells which may play a role in prostatic carcinogenesis.

Transforming growth factor beta as a clinical biomarker for prostate cancer.

OBJECTIVES: Tumor biomarkers to detect prostate cancer earlier may reduce prostate cancer deaths. Transforming growth factor-beta1 and -beta2 (TGF-beta1 and -beta2) become overexpressed in prostate cancer and might be useful tumor markers of prostate cancer. METHODS: Plasma and urinary TGF-beta1 and plasma TGF-beta2 levels were studied preoperatively in 74 consecutive patients who had prostate cancer and underwent radical prostatectomy and were compared with those of 29 similarly aged male control patients who had no clinical evidence of prostate cancer. RESULTS: Plasma TGF-beta1 levels were similar in both prostate cancer and control groups and did not correlate with serum prostate-specific antigen (PSA), clinical and pathologic stages, or Gleason grade. Urinary TGF-beta1 levels, however, increased 3.5-fold in patients with prostate cancer relative to controls and tended to be higher with advancing clinical and pathologic stages. Plasma TGF-beta2 levels, like plasma TGF-beta1 levels, were similar for both the study and control groups, but when stratified by pathologic stage or Gleason grade, patients with prostate cancer with pathologic Stage T2a and Gleason grade of 3 or less had significantly increased plasma TGF-beta2 levels as compared with either control patients or patients with prostate cancer with pathologic Stages T2b/T2c and T3/T4 or Gleason grade of 4 or more, suggesting that early prostate cancer may contribute to plasma TGF-beta2 levels. CONCLUSIONS: Unlike plasma TGF-beta1 levels, urinary TGF-beta1 and plasma TGF-beta2 levels were higher in patients with prostate cancer and may be useful biomarkers of prostate cancer.

Can prostate-specific antigen be used as a valid end point to determine the efficacy of chemotherapy for advanced prostate cancer?

It is current practice in many clinical trials evaluating new chemotherapy regimens for the treatment of advanced prostate cancer to use prostate-specific antigen (PSA) decline as a response criteria with the assumption that the level of PSA reflects the efficacy of chemotherapy. Advanced prostate cancer is heterogeneous; therefore, the validity of PSA decline as a measurable end point was studied in advanced human prostate-cancer cell lines: androgen-sensitive LNCaP and androgen-insensitive PC3 cells. Each cell line was grown for 4 days with escalating doses of Adriamycin or vinblastine. Cell counts, intracellular PSA concentrations, and secreted PSA levels were determined daily for 4 days. Untreated LNCaP cells had constant secretion of PSA per cell. In contrast, LNCaP cells treated with Adriamycin or vinblastine had an 80% reduction in cell numbers and a 3-fold increase in secreted PSA per cell by day 4. In contrast, PC3 cells had a different response to Adriamycin and vinblastine. Both drugs reduced cell numbers by 97% of control values and suppressed PSA production in the remaining viable cells by 4 days in culture. Thus, prostate-cancer cell production of PSA is variable with chemotherapy and the PSA level may not accurately reflect the actual tumor response to chemotherapy.

Bone morphogenetic protein-6 expression in normal and malignant prostate.

Bone morphogenetic proteins (BMPs) have multiple biologic functions, including bone formation and embryonic induction. One of these proteins, BMP-6, was reportedly expressed at high levels in human prostate cancers that had also metastasized to bone. This study investigated both BMP-6 mRNA and protein expression in normal and malignant rat and human prostate tissues. BMP-6 was detected in both rat normal prostate and in Dunning rat-prostate adenocarcinoma sublines. The levels of BMP-6 mRNA and protein were similar for normal and malignant rat prostate, regardless of the metastatic potential. Moreover, castration had no apparent effect on BMP-6 production in rat normal ventral prostate, suggesting an androgen-independent gene regulation of this protein. BMP-6 mRNA and protein were also produced by normal and neoplastic human prostate cancer (radical prostatectomy specimens and human carcinoma cell lines DU145 and PC3). BMP-6 mRNA and protein expression, however, was higher in prostate cancer as compared with adjacent normal prostate, with higher-grade tumors (Gleason score of 6 or more) having greater BMP-6 immunostaining than the lower-grade tumors (Gleason score of 4 or less). Taken together, these results suggest that BMP-6 protein expression may serve as a potential marker for prostate cancer but not as a metastatic marker. Moreover, BMP-6 may contribute to prostate neoplastic behavior even in the absence of androgens.

Prostate cancer cells lose their sensitivity to TGFßI growth inhibition with tumor progression.

The observation that advanced prostate cancer has reduced sensitivity to transforming growth factor ßI (TGFßI) growth inhibition suggests that the acquisition of TGFßI resistance may play a role in prostate tumor progression. Using the Dunning R3327 rat prostate adenocarcinoma model, it was determined that prostate carcinoma cells became less responsive to TGFßI growth inhibition with differentiated tumors more resistant to TGFßI. A TGFß receptor defect was not found in advanced prostate carcinoma cells because both the type I and II TGFß receptors were present and functional. Moreover, TGFα/epidermal growth factor receptor (EGFR) and basic fibroblast growth factor (bFGF)/fibroblast growth factor receptor (FGFR) autocrine stimulatory pathways, which may potentially counter-regulate the inhibitory effects of TGFßI, were not present with prostate cancer progression. However, the likelihood that other prereceptor stimulatory pathways or TGFß postreceptor signaling alterations are responsible for reduced sensitivity to TGFßI growth inhibition remains to be elucidated.

Actions of the testicular paracrine factor (P-Mod-S) on Sertoli cell transferrin secretion throughout pubertal development.

Peritubular cells that surround the seminiferous tubules have been shown to produce a paracrine factor, termed P-Mod-S, that has dramatic effects on Sertoli cell function in vitro and is postulated to be important in the control of testicular function. The current study was designed to determine whether P-Mod-S has the ability to regulate Sertoli cell function during pubertal development. Sertoli cells were isolated from 10-, 20-, and 35-day-old rats which correspond to the prepubertal, mid-pubertal, and late pubertal stages of development. Histochemical analysis of cultured cells isolated from each age group was performed to establish the purity of the cell populations used. Testicular transferrin production by Sertoli cells was used as a marker of cellular differentiation. Basal production of transferrin by the cultured cells was found to increase during the pubertal period. P-Mod-S stimulated transferrin production by Sertoli cells isolated from 10-, 20-, and 35-day-old rats. FSH appears to enhance the ability of Sertoli cells to respond to P-Mod-S with cells obtained from 10-day-old rats. Sertoli cells from 35-day-old rats were nonresponsive to regulatory agents such as FSH. P-Mod-S alone, however, significantly stimulated transferrin production by Sertoli cells from this more adult stage of development. P-Mod-S was the only individual regulatory agent tested that could stimulate transferrin production by Sertoli cells from 35-day-old rats. Results indicate that P-Mod-S has the ability to regulate Sertoli cell function throughout pubertal development. Observations suggest that P-Mod-S and FSH may act together in the prepubertal testis to promote Sertoli cell differentiation and that P-Mod-S may act in the adult testis to maintain optimal Sertoli cell function and differentiation.

Cell-cell interactions and the regulation of testis function.

Regulatory interactions have been shown to occur between all the testicular cell types considered. The paracrine factors mediating these interactions generally influence either cellular growth or differentiation. The regulation of cellular growth is essential in the developing testis and is required for the maintenance of spermatogenesis in the adult testis. The rapid rate of germinal cell proliferation and the continuous but slowed growth of the peritubular cells and Leydig cells requires the presence of specific growth factors in the adult. Therefore, cell-cell interactions have evolved that involve growth factors such as IGF, TGF-alpha, TGF-beta and NGF. Other growth factors such as FGF or less characterized components like the seminiferous growth factor (SGF) also may be involved in the paracrine regulation of testis cell growth. An alternate cellular parameter to cell growth to consider is the regulation of cellular function and differentiation. A number of endocrine agents and locally produced paracrine factors have been shown to control and maintain testis cell function and differentiation. Cell-cell interactions mediated by factors such as androgens, POMC peptides, and PModS are all primarily directed at the regulation of cellular differentiation. Therefore, the agents which mediate cell-cell interactions in the testis can generally be categorized into factors that regulate cell growth or those which influence cellular differentiation. The specific cell-cell interactions identified will likely be the first of a large number of cellular interactions yet to be investigated. Although a number of potentially important cell-cell interactions have been identified, future research will require the elucidation of the in vivo physiological significance of these interactions. The existence of different cell types and potential cell-cell interactions in a tissue implies that the actions of an endocrine agent on a tissue will not simply involve a single hormone and single cell. The endocrine regulation of testis function will have effects on cell-cell interactions and be affected by local cell-cell interactions. The ability of LH to influence Leydig cell androgen production promotes a cascade of interactions mediated through several cell types to maintain the process of spermatogenesis. FSH actions on Sertoli cells also promote cell-cell interactions that influence germinal cell development, peritubular myoid cell differentiation and Leydig cell function. Therefore, elucidation of the endocrine regulation of testis function requires an understanding of the local cell-cell interactions in the testis.

Analysis of the androgen receptor in isolated testicular cell types with a microassay that uses an affinity ligand.

A microassay for the androgen receptor was developed to investigate the cellular distribution of receptor in freshly isolated testicular cell types. The microassay uses an androgen affinity ligand, 17 beta-dihydrotestosterone bromoacetate. Binding of this ligand by the androgen receptor is rapid and irreversible, which permits the development of a highly sensitive assay. The androgen receptor microassay is completed within 4 h and detects receptor in as little as 0.5 micrograms cellular protein. There was no detectable binding of the affinity label by albumin or Sertoli cell-secreted proteins, including androgen-binding protein. Androgen receptor was found in cellular sonicates of human foreskin fibroblast, rat ventral prostate, rat kidney, and rat liver. Although the relative distribution of receptor was similar to that obtained using a traditional equilibrium binding assay, the levels of receptor were significantly higher using the microassay. The androgen receptor microassay was subsequently used to investigate the receptor in isolated testicular cell types. Androgen receptor was detected in freshly isolated peritubular myoid cells (80 fmol/micrograms DNA), Sertoli cells (88 fmol/micrograms DNA), and Leydig cells (35 fmol/micrograms DNA). No androgen receptor was detected in a mixed population of germ cells. Hormones were not found to influence androgen receptor levels in cultured peritubular cells or Sertoli cells. Electrophoretic analysis of androgen receptor radiolabeled with the affinity ligand demonstrates a single 52-kDa form of the receptor in peritubular cells, Sertoli cells, and Leydig cells. The size of the androgen receptor species detected in the rat testicular cell types was slightly smaller than the 56-kDa protein detected in a human fibroblast cell line. The current study demonstrates the utility of the microassay and affinity ligand to investigate androgen receptor biology. Data indicate that androgen receptors are present in several testicular cell types and suggest that the control of testicular function by androgens probably involves actions on multiple cell types.