Paucity of PD-L1 expression in prostate cancer: innate and adaptive immune resistance.

BACKGROUND: Primary prostate cancers are infiltrated with programmed death-1 (PD-1) expressing CD8+ T-cells. However, in early clinical trials, men with metastatic castrate-resistant prostate cancer did not respond to PD-1 blockade as a monotherapy. One explanation for this unresponsiveness could be that prostate tumors generally do not express programmed death ligand-1 (PD-L1), the primary ligand for PD-1. However, lack of PD-L1 expression in prostate cancer would be surprising, given that phosphatase and tensin homolog (PTEN) loss is relatively common in prostate cancer and several studies have shown that PTEN loss correlates with PD-L1 upregulation--constituting a mechanism of innate immune resistance. This study tested whether prostate cancer cells were capable of expressing PD-L1, and whether the rare PD-L1 expression that occurs in human specimens correlates with PTEN loss. METHODS: Human prostate cancer cell lines were evaluated for PD-L1 expression and loss of PTEN by flow cytometry and western blotting, respectively. Immunohistochemical (IHC) staining for PTEN was correlated with PD-L1 IHC using a series of resected human prostate cancer samples. RESULTS: In vitro, many prostate cancer cell lines upregulated PD-L1 expression in response to inflammatory cytokines, consistent with adaptive immune resistance. In these cell lines, no association between PTEN loss and PD-L1 expression was apparent. In primary prostate tumors, PD-L1 expression was rare, and was not associated with PTEN loss. CONCLUSIONS: These studies show that some prostate cancer cell lines are capable of expressing PD-L1. However, in human prostate cancer, PTEN loss is not associated with PD-L1 expression, arguing against innate immune resistance as a mechanism that mitigates antitumor immune responses in this disease.

Prevalence of BPH and lower urinary tract symptoms in West Africans.

BACKGROUND: BPH and lower urinary tract symptoms (LUTS) are very common among older men in Western countries. However, the prevalence of these two conditions in the developing countries is less clear. METHODS: We assessed the age-standardized prevalence of BPH and/or LUTS among West Africans in a probability sample of 950 men aged 50-74 in Accra, Ghana, with no evidence of biopsy-confirmed prostate cancer after screening with PSA and digital rectal examination (DRE). Information on LUTS was based on self-reports of the International Prostate Symptom Score (IPSS). BPH was estimated using DRE, PSA levels and imputed prostate volume. RESULTS: The prevalence of DRE-detected enlarged prostate was 62.3%, while that of PSA≥1.5 ng ml(-1) (an estimate of prostate volume ≥ 30 cm(3)) was 35.3%. The prevalence of moderate-to-severe LUTS (IPSS≥8) was 19.9%. The prevalence of IPSS≥8 and an enlarged prostate on DRE was 13.3%. Although there is no universally agreed-upon definition of BPH/LUTS, making comparisons across populations difficult, BPH and/or LUTS appear to be quite common among older Ghanaian men. CONCLUSIONS: We found that after age standardization, the prevalence of DRE-detected enlarged prostate in Ghanaian men is higher than previously reported for American men, but the prevalence of LUTS was lower than previously reported for African Americans. Further studies are needed to confirm these findings and identify the risk factors for BPH in both Africans and African Americans.

8q24 allelic imbalance and MYC gene copy number in primary prostate cancer.

Four independent regions within 8q24 near the MYC gene are associated with risk for prostate cancer (Pca). Here, we investigated allelic imbalance (AI) at 8q24 risk variants and MYC gene DNA copy number (CN) in 27 primary Pcas. Heterozygotes were observed in 24 of 27 patients at one or more 8q24 markers and 27% of the loci exhibited AI in tumor DNA. The 8q24 risk alleles were preferentially favored in the tumors. Increased MYC gene CN was observed in 33% of tumors, and the co-existence of increased MYC gene CN with AI at risk loci was observed in 86% (P<0.004 exact binomial test) of the informative tumors. No AI was observed in tumors, which did not reveal increased MYC gene CN. Higher Gleason score was associated with tumors exhibiting AI (P=0.04) and also with increased MYC gene CN (P=0.02). Our results suggest that AI at 8q24 and increased MYC gene CN may both be related to high Gleason score in Pca. Our findings also suggest that these two somatic alterations may be due to the same preferential chromosomal duplication event during prostate tumorigenesis.

Early prostate cancer antigen expression in predicting presence of prostate cancer in men with histologically negative biopsies.

PURPOSE: Early prostate cancer antigen is a nuclear matrix protein that was recently shown to be expressed in prostate adenocarcinoma and adjacent benign tissue. Previous studies have demonstrated early prostate cancer antigen expression in benign prostate tissue up to 5 years before a diagnosis of prostate carcinoma, suggesting that early prostate cancer antigen could be used as a potential predictive marker. MATERIALS AND METHODS: We evaluated early prostate cancer antigen expression by immunohistochemistry using a polyclonal antibody (Onconome Inc., Seattle, Washington) on benign biopsies from 98 patients. Biopsies were obtained from 4 groups that included 39 patients with first time negative biopsy (group 1), 24 patients with persistently negative biopsies (group 2), 8 patients with initially negative biopsies who were subsequently diagnosed with prostate carcinoma (group 3) and negative biopsies obtained from 27 cases where other concurrent biopsies contained prostate carcinoma (group 4). Early prostate cancer antigen staining was assessed by 2 of the authors who were blind to the group of the examined sections. Staining intensity (range 0 to 3) and extent (range 1 to 3) scores were assigned. The presence of intensity 3 staining in any of the blocks of a biopsy specimen was considered as positive for early prostate cancer antigen for the primary outcome in the statistical analysis. In addition, as secondary outcomes we evaluated the data using the proportion of blocks with intensity 3 early prostate cancer antigen staining, the mean of the product of staining intensity and staining extent of all blocks within a biopsy, and the mean of the product of intensity 3 staining and extent. RESULTS: Primary outcome analysis revealed the proportion of early prostate cancer antigen positivity to be highest in group 3 (6 of 8, 75%) and lowest in group 2 (7 of 24, 29%, p=0.04 for differences among groups). A relatively higher than expected proportion of early prostate cancer antigen positivity was present in group 1 (23 of 39, 59%). Early prostate cancer antigen was negative in 41% of group 4 who were known to harbor prostate carcinoma. The proportion of early prostate cancer antigen positivity was statistically significantly lower in group 2 than in each of the other groups when compared pairwise. A lower proportion of early prostate cancer antigen positivity was encountered in older archival tissue blocks (p<0.0001) pointing to a potential confounding factor. Corrected for block age, group 3 was the only group to remain statistically significantly different in early prostate cancer antigen positivity compared to the reference group 2. Similar findings were obtained when adjustments for patient age were made and when analysis was based on secondary outcome measurements. CONCLUSIONS: Our study showed a higher proportion of early prostate cancer antigen expression in initial negative prostate biopsy of patients who were diagnosed with prostate carcinoma on subsequent followup biopsies. We found a relatively high proportion of early prostate cancer antigen positivity (59%) in the group with first time negative biopsies and a potential 41% rate of false-negative early prostate cancer antigen staining in benign biopsies from cases with documented prostate carcinoma on concurrent cores. The lower early prostate cancer antigen positivity in cases with older blocks raises the question of a confounding effect of block age. Additional studies on the antigenic properties of early prostate cancer antigen in archival material are required to further delineate the usefulness of early prostate cancer antigen immunostaining on biopsy material.

Expression of the DNA-PK binding protein E4-34K fails to confer radiation sensitivity to mammalian cells.

PURPOSE: The adenovirus E4orf6 34 kDa protein (E4-34k) is known to disrupt V(D)J recombination as a result of its interaction with the catalytic subunit of cellular DNA-dependent protein kinase (DNA-PK(cs)), a major participant in the repair of DNA double-strand breaks (DSB). Previous studies have shown that cells with disrupted DSB repair and V(D)J recombination due to attenuation of DNA-PK(cs) activity exhibit a radiation-sensitive phenotype. It is not known at present whether the E4-34k protein can also modify cellular response to ionizing radiation. In an attempt to develop a novel gene therapy strategy to modify cellular radiation response, we sought to determine if expression of the adenovirus E4-34k protein resulted in sensitization to clinically relevant doses of ionizing radiation. MATERIALS AND METHODS: In order to minimize potential bias resulting from selection procedures, we performed clonogenic survival assays on DU 145 prostate cancer cells, RKO colorectal cancer cells and 293 kidney cells following transient transfection of E4-34k- and/or E1B-55k-expressing plasmids. Western blots and immunohistochemical analyses were used to demonstrate E4-34k expression within transfected cells. FACS sorting was carried out to enrich cells transfected with a plasmid that expresses both E4-34k and enhanced green fluorescent protein. RESULTS: It is shown that E4-34k expression does not affect cellular radiosensitivity of transiently transfected populations of either DU 145 prostate or RKO colon cancer cell lines. Similarly, the radiosensitivity of human embryonic kidney 293 cells, which constitutively express the E1B-55k protein, was also unaffected. The radiosensitivity of DU 145 cells co-transfected with E4-34k- and E1-55K-expressing plasmids was unchanged, suggesting that the adenovirus E1B-55k protein does not augment any effects E4-34k might have on DNA-PK(cs) activity. CONCLUSIONS: The lack of radiosensitization by E4-34k expression is quite intriguing as it is known that E4-34k interaction with DNA-PK(cs) causes disruption of V(D)J recombination, a process dependent on DSB rejoining. These data suggest that for future studies, preferential targeting of DNA-PK(cs) DSB activity will be required to influence cellular radiosensitivity.

A phase I trial of CV706, a replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy.

CV706 is a prostate-specific antigen (PSA)-selective, replication-competent adenovirus that has been shown to selectively kill human prostate cancer xenografts in preclinical models. To study the safety and activity of intraprostatic delivery of CV706, a Phase I dose-ranging study for the treatment of patients with locally recurrent prostate cancer after radiation therapy was conducted. Twenty patients in five groups were treated with between 1 x 10(11) and 1 x 10(13) viral particles delivered by a real-time, transrectal ultrasound-guided transperineal technique using a three-dimensional plan. The primary end point was the determination of treatment-related toxicity. Secondary objectives included evaluation of the antitumor activity of CV706 and monitoring for other correlates of antineoplastic action. In this study, CV706 was found to be safe and was not associated with irreversible grade 3 or any grade 4 toxicity. No grade >1 alterations in liver function tests associated with CV706 administration were observed. Posttreatment prostatic biopsies and detection of a delayed "peak" of circulating copies of virus provided evidence of intraprostatic replication of CV706. The study defined the timing of CV706 shedding into blood and urine as well as the appearance of circulating Ad5 neutralizing antibodies. Finally, this study documents the serum PSA response of treated patients and reveals a dose response showing that all five patients who achieved a > or =50% reduction in PSA were treated with the highest two doses of CV706. This study represents the first clinical translation of a prostate-specific, replication-restricted adenovirus for the treatment of prostate cancer. Taken together, this study documents that intraprostatic delivery of CV706 can be safely administered to patients, even at high doses, and the data also suggest that CV706 possesses enough clinical activity, as reflected by changes in serum PSA, to warrant additional clinical and laboratory investigation.

Rational basis for Trk inhibition therapy for prostate cancer.

BACKGROUND: Prostatic cancer cells are lethal because they acquire the ability to activate survival pathways that do not require androgenic stimulation. As a rational approach to developing effective therapy for these devastating cells, specific signal transduction pathways uniquely required for the survival of these nonandrogen-dependent prostate cancer cells must be identified. Previous studies suggested that the neurotrophin/trk signal transduction axis may regulate such unique survival pathways. In the present study, the changes in expression of the neurotrophins (NGF, BDNF, and NT-3) and their cognate receptors (i.e., trk and p75NTR) during the progression of normal prostatic epithelial cells to malignancy were documented. Additionally, the consequences of inhibiting these trk signaling pathways on the in vitro survival of prostate cancer cells was tested. METHODS: Immmunocytochemistry, RT-PCR, and ELISA assays were used to characterize the changes in the neurotrophin ligands (i.e., NGF, BDNF, and NT-3) and their cognate high-affinity (i.e., trk A, B, and C) and low-affinity neurotrophin (i.e., p75 NTR) receptors in normal vs. malignant human prostatic tissues. CEP-751 is an indolocarbazole compound specifically designed to inhibit the initiation of these neurotrophin/trk signal transductions. The consequence of CEP-751 inhibition of trk signaling for in vitro clonogenic survival of a series of human prostatic cancer lines was also tested. RESULTS: These studies demonstrated that normal prostatic tissue from patients without prostate cancer contains substantial levels of nerve growth factor (NGF), which is produced in a paracrine manner by stromal cells. These stromal cells lack both trk and p75NTR receptors. In contrast, normal prostatic epithelial cells from patients without prostate cancer do not secrete detectable levels of neurotrophins, but do express trk A and p75 NTR. While the NGF/trkA/p75 NTR axis is present in the normal prostate, normal prostatic epithelial cells do not depend on this axis for their survival. In contrast, malignant prostate epithelial cells directly secrete a series of neurotrophins (i.e., NGF, BDNF, and/or NT-3) and express at least one if not more of the trk receptor proteins (i.e., trk A, B, and/or C), while no longer expressing the p75NTR receptors. In addition, inhibition of autocrine trk signaling via CEP-751 treatment induces the apoptotic death of these malignant cells. CONCLUSIONS: Prostate carcinogenesis involves molecular changes leading to the paracrine and/or autocrine production of a series of neurotrophins. This is coupled to the ectopic expression of trk B and trk C, as well as to the continued expression of trk A, and the loss of expression of p75NTR receptors. These changes result in the acquisition by malignant prostate cells of a unique requirement for trk signaling pathways for survival. Based on these findings, trk inhibition is a novel, rational approach for prostate cancer therapy.

Cancer cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer as vaccines for the treatment of genitourinary malignancies.

When irradiated and administered intradermally as vaccines, cancer cells engineered to secrete high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) by gene transfer elicit potent anticancer immune responses in a variety of animal tumor models. Upon vaccination, antigens present in the cancer cells are phagocytosed and processed by skin dendritic cells. These dendritic cells then prime anticancer immune responses by presenting antigenic peptides to T cells. The immune responses generated are capable of eradicating small but lethal cancer cell inocula with minimal toxicity in preclinical animal tumor studies. To develop this vaccination strategy for the treatment of human genitourinary cancers, we have conducted phase I clinical trials using human genitourinary cancer cells as sources of cancer cell antigens. In the first human clinical trial of genetically engineered cancer cell vaccines, a phase I clinical trial of kidney cancer cell vaccines (n = 18), kidney cancer cells were removed at surgery, propagated briefly in vitro, and then genetically modified to secrete high levels of GM-CSF via ex vivo transduction with the retrovirus MFG-GM-CSF. After irradiation, the kidney cancer cells were administered as vaccines to 18 patients with advanced kidney cancers. Vaccine treatment, which caused few side effects, nonetheless appeared to trigger anticancer immune responses manifest as conversion of delayed-type hypersensitivity (DTH) skin responses against irradiated autologous cancer cells after vaccination. Biopsies of vaccine sites yielded findings reminiscent of biopsies from preclinical animal model studies, with evidence of vaccine cell recruitment of dendritic cells, T cells, and eosinophils. One patient with measurable kidney cancer metastases treated at the highest vaccine dose level experienced a partial treatment response. The bioactivity of GM-CSF-secreting autologous cancer cell vaccines was confirmed in a phase I clinical trial for prostate cancer (n = 8). Vaccine cells were prepared from surgically harvested prostate tumors by ex vivo transduction with MFG-GM-CSF in a manner similar to that used for the kidney cancer trial. Vaccine treatment was well tolerated and associated with induction of anticancer immunity as assessed using DTH skin testing. In addition, new antiprostate cancer cell antibodies were detected in serum samples from treated men as a consequence of vaccination. These first clinical trials of GM-CSF-secreting cancer cell vaccines for the treatment of genitourinary cancers have demonstrated both safety and bioactivity, in that very few side effects have been seen and anticancer immune responses have been detected. Future clinical studies will be required to assess vaccine treatment efficacy, refine vaccination dose and schedule, define the appropriate clinical context for the use of such vaccines, and ascertain optimal combinations involving vaccines and other local or systemic anticancer treatments.

Genetic alterations in urinary bladder carcinosarcoma: evidence of a common clonal origin.

The cellular origin of carcinosarcoma of the bladder is unknown. We addressed this issue by using microsatellite analysis for loss of heterozygosity (LOH) in both the carcinomatous and sarcomatous components of 6 bladder tumors. We tested 40 microsatellite markers from 19 human chromosomes and compared the genetic alterations between the two separately isolated components. The potential relevance of the E-cadherin pathway was also evaluated by immunohistochemistry. All 6 cases revealed identical LOH on chromosomal arms 9p, 9q, 8p, and 8q, corresponding to relatively early events in bladder carcinogenesis. Discordant losses between two alleles in the remaining chromosomes, associated with progression, were seen in all tumors with a trend toward a higher incidence in the more advanced tumors (N1M1 and N1Mx). E-cadherin was strongly expressed in the carcinomatous components (5 of 6), whereas most of sarcomatous elements displayed absence of the protein product (4 of 6). These results indicate that both the carcinomatous and sarcomatous components of carcinosarcoma are derived from a common stem cell. Downregulation of E-cadherin may define one of the pathways responsible for conversion of epithelial cells to the sarcomatous phenotype.

Induction of immunity to prostate cancer antigens: results of a clinical trial of vaccination with irradiated autologous prostate tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer.

Vaccination with irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting gene-transduced cancer vaccines induces tumoricidal immune responses. In a Phase I human gene therapy trial, eight immunocompetent prostate cancer (PCA) patients were treated with autologous, GM-CSF-secreting, irradiated tumor vaccines prepared from ex vivo retroviral transduction of surgically harvested cells. Expansion of primary cultures of autologous vaccine cells was successful to meet trial specifications in 8 of 11 cases (73%); the yields of the primary culture cell limited the number of courses of vaccination. Side effects were pruritus, erythema, and swelling at vaccination sites. Vaccine site biopsies manifested infiltrates of dendritic cells and macrophages among prostate tumor vaccine cells. Vaccination activated new T-cell and B-cell immune responses against PCA antigens. T-cell responses, evaluated by assessing delayed-type hypersensitivity (DTH) reactions against untransduced autologous tumor cells, were evident in two of eight patients before vaccination and in seven of eight patients after treatment. Reactive DTH site biopsies manifested infiltrates of effector cells consisting of CD45RO+ T-cells, and degranulating eosinophils consistent with activation of both Th1 and Th2 T-cell responses. A distinctive eosinophilic vasculitis was evident near autologous tumor cells at vaccine sites, and at DTH sites. B-cell responses were also induced. Sera from three of eight vaccinated men contained new antibodies recognizing polypeptides of 26, 31, and 150 kDa in protein extracts from prostate cells. The 150-kDa polypeptide was expressed by LNCaP and PC-3 PCA cells, as well as by normal prostate epithelial cells, but not by prostate stromal cells. No antibodies against prostate-specific antigen were detected. These data suggest that both T-cell and B-cell immune responses to human PCA can be generated by treatment with irradiated, GM-CSF gene-transduced PCA vaccines.

Digital dynamic telepathology--the Virtual Microscope.

The Virtual Microscope is being designed as an integrated computer hardware and software system that generates a highly realistic digital simulation of analog, mechanical light microscopy. We present our work over the past year in meeting the challenges in building such a system. The enhancements we made are discussed, as well as the planned future improvements. Performance results are provided showing the system scales well, so that many users can be adequately serviced by an appropriately configured data server.

The Virtual Microscope.

We present the design of the Virtual Microscope, a software system employing a client/server architecture to provide a realistic emulation of a high power light microscope. We discuss several technical challenges related to providing the performance necessary to achieve rapid response time, mainly in dealing with the enormous amounts of data (tens to hundreds of gigabytes per slide) that must be retrieved from secondary storage and processed. To effectively implement the data server, the system design relies on the computational power and high I/O throughput available from an appropriately configured parallel computer.

Progesterone receptor and the mechanism of action of progesterone antagonists.

Currently available progesterone antagonists have been suggested to fall into two categories based on differences in how they interact with and inactivate the progesterone receptor (PR). The anti-progestin ZK98299 (Type I) impairs PR association with DNA, while Type II compounds (RU486, ZK112993, ZK98734) promote PR binding to DNA. Type II agents, therefore, appear to inhibit receptor activity at a step downstream of DNA binding, presumably failing to induce conformational changes in PR structure requird for enhancement of transcription. This paper discusses both published and unpublished data supporting the concept of two types of progestin antagonists. Using PR-mediated induction of reporter genes in breast cancer cells as an assay for biological response, both types of anti-progestins, after correction for difference in steroid binding affinity, inhibit progestin induction substoichiometrically. However, Type II anti-progestins are more potent, inhibiting at lower ratios of antagonist to agonist than ZK98299. This suggests that in addition to behaving by classical competitive mechanisms these compounds (in particular Type II) may exhibit additional activity as transrepressors of PR in the same cell bound to hormone agonist. Transrepression may occur by the combined mechanisms of heterodimerization and competition for binding to DNA. In support of this, mixed ligand dimers form readily in solution between a PR subunit bound to agonist and another bound to either type of anti-progestin, whereas these mixed ligand dimers bind poorly, if at all, to specific progesterone response elements (PREs) in vitro. Additionally, when added as a single ligand, Type II agents increase PR dimerization in solution and PR affinity for PREs as compared with single ligand dimers formed by progestin agonist. This contrasts with ZK98299, when given as a single ligand, which reduces PR affinity for PREs without disrupting solution dimerization. Thus the higher affinity of PR for PREs may account for the greater biological potency of Type II compounds as compared with ZK98299. As a further distinction between types of antiprogestins, ZK98299 minimally stimulates phosphorylation of PR whereas RU486 increases site-specific phosphorylation of PR in a manner indistinguishable from that of hormone agonist. Additionally, ZK98299 is not susceptible in vivo to functional switching to a partial agonist by cross talk with cAMP signal transduction pathways, as occurs with Type II compounds. Thus, ZK98299 under certain conditions may be a more pure antagonist than Type II compounds.

Effects of the steroid antagonist RU486 on dimerization of the human progesterone receptor.

We previously reported, using a coimmunoprecipitation assay, that the B form (PR-B) of the human progesterone receptor from T47D human breast cancer cells dimerizes in solution with the A receptor (PR-A) and that the extent of dimerization correlates with receptor binding activity for specific DNA sequences [DeMarzo, A.M., Beck, C.A., Oñate, S.A., & Edwards, D.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 72-76]. This suggested that solution dimerization is an intermediate step in the receptor activation process. The present study has tested the effects of the progesterone antagonist RU486 on solution dimerization of progesterone receptors (PR). As determined by the coimmunoprecipitation assay, RU486 binding did not impair dimerization of receptors; rather, the antagonist promoted more efficient solution dimerization than the progestin agonist R5020. This enhanced receptor dimerization correlated with a higher DNA binding activity for transformed receptors bound with RU486. RU486 has been shown previously to produce two other alterations in the human PR when compared with R5020. PR-RU486 complexes in solution exhibit a faster sedimentation rate (6 S) on salt-containing sucrose density gradients than PR-R5020 complexes (4 S), and PR-DNA complexes have a faster electrophoretic mobility on gel-shift assays in the presence of RU486. We presently show that the 6 S PR-RU486 complex is a receptor monomer, not a dimer. The increased sedimentation rate and increased mobility on gel-shift assays promoted by RU486 were also observed with recombinant PR-A and PR-B separately expressed in insect cells from baculovirus vectors. These results suggest that RU486 induces a distinct conformational change both in PR monomers in solution and in dimers bound to DNA. We also examined whether conformational changes in PR induced by RU486 would prevent a PR polypeptide bound to RU486 from heterodimerization with another PR polypeptide bound to R5020. To evaluate this, PR-A and PR-B that were separately bound to R5020 or RU486 in whole cells were mixed in vitro. PR-A-RU486 was capable of dimerization with PR-B-R5020, and this was demonstrated for heterodimers both formed in solution and bound to specific DNA. The capability to form heterodimers in vitro raises the possibility that the antagonist action of RU486 in vivo could in part be imposed in a dominant negative fashion through heterodimerization between one receptor subunit bound to an agonist and another bound to RU486.

Characterization and functional properties of the A and B forms of human progesterone receptors synthesized in a baculovirus system.

Human progesterone receptors (PR) were overexpressed in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus system. Recombinant viruses were constructed that produced either full-length A (94K) or B (120K) forms of human PR, and each was expressed as a functional protein. Steroid and DNA binding activities were found to be indistinguishable from that of endogenous human PR in T47D breast cancer cells. Moreover, as analyzed by gel-mobility shift, recombinant PR-A and PR-B each bound to specific progesterone response elements in a strictly hormone-dependent manner. Native receptors expressed in Sf9 cells also exhibited structural properties similar to that of endogenous PR. Cytosolic PR (PR-A or PR-B), prepared in low salt buffer, sedimented on density gradients as an 8S oligomeric complex that was converted largely to 4S by treatment with 0.4 M NaCl. Immune isolation of the 8S cytosol PR complex and analysis of protein composition revealed the presence of two specific copurifying proteins of approximately 90K and 70K. The 90-K component was identified immunologically as heat shock protein 90. The 70-K component was not identified but is likely to be the insect equivalent of heat shock protein 70. Immune isolation of PR from Sf9 cells metabolically labeled with [32Pi], revealed that expressed PR was capable of being phosphorylated in insect cells. Hormone addition to Sf9 cells, however, did not stimulate the same increase in PR phosphorylation or upshift in mobility on sodium dodecyl sulfate gels that occurs with endogenous receptors in T47D cells. Thus some, but not all, phosphorylations occur with human PR expressed in Sf9 cells. These phosphorylation data, together with the fact that expressed PR required hormone for DNA binding, indicate that the hormone-dependent phosphorylation step responsible for PR upshifts on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is not required for receptor binding to DNA. The baculovirus expression system, therefore, may prove valuable in dissecting the functional role(s) for both hormone-dependent and hormone-independent PR phosphorylation.

Mechanisms controlling steroid receptor binding to specific DNA sequences.

Mammalian progesterone receptors activated by hormone binding in nuclei of intact cells exhibit substantially higher binding activity for specific DNA sequences than receptors bound with hormone and activated in cell-free cytosol. Differences in DNA-binding activity occur despite the fact that both activated receptor forms sediment at 4S on sucrose gradients and are apparently dissociated from the heat shock protein 90. This suggests that hormone-induced release of heat shock protein 90 from receptors is necessary, but not sufficient for maximal activation of DNA binding. This report is a review of studies from our laboratories that have examined the role of receptor interaction with other nuclear protein factor(s), and receptor dimerization in solution, as additional regulatory steps involved in the process of receptor activation and binding to specific gene sequences.

Dimerization of mammalian progesterone receptors occurs in the absence of DNA and is related to the release of the 90-kDa heat shock protein.

In this study we have demonstrated that dimerization of mammalian progesterone receptors (PR) occurs in the absence of DNA. A specific immune coisolation assay was performed on extracts of T-47D human breast cancer cells with a monoclonal antibody specific for the full-length B form of progesterone receptor (PR-B). This resulted in coisolation of significant amounts of truncated form-A receptors (PR-A), indicating the presence of stable PR-A.PR-B dimers in solution. A positive correlation was observed between the ability of different receptor forms to oligomerize in solution and their ability to bind to specific DNA sequences. The ability to form stable PR-A.PR-B oligomers in the absence of DNA was also found to correlate with release of 90-kDa heat shock protein (hsp90) from the unactivated PR complex. These results support the hypothesis that dimerization in the absence of DNA is an important mechanism controlling receptor DNA-binding function and that hsp90 release may be a key step regulating dimerization. This suggests that hsp90 may function to repress DNA-binding activity indirectly by blocking receptor dimerization.

Ultrastructural localization of matrix vesicles and alkaline phosphatase in the Swarm rat chondrosarcoma: their role in cartilage calcification.

Although it is generally acknowledged that matrix vesicles and alkaline phosphatase are required for cartilage calcification, their precise role in initiation or propagation of mineralization remains controversial. We have investigated this problem in a neoplastic model of chondrogenesis, the Swarm rat chondrosarcoma. During serial transplantation these malignant chondrocytes fail to undergo terminal differentiation and do not calcify. To facilitate the study of maturational events in this neoplasm we have developed an intraperitoneal model which grows as discrete free floating tumor nodules. The youngest cells are those on the exterior and maturation progresses towards the center. Ultrastructural examination of the tumor reveals the presence of numerous matrix vesicles in the interterritorial matrix of immature and mature cells. Histochemical localization of alkaline phosphatase reveals a distribution of enzyme activity which varies with the state of maturation of the cells. The most immature cells express large amounts of enzyme on their plasmalemma and in inter-territorial matrix vesicles. The older cells lose membrane activity but maintain activity in their matrix vesicles. These results suggest that while matrix vesicles and/or alkaline phosphatase may be necessary for calcification to proceed, their mere presence is by itself insufficient to initiate or maintain the process.