Expression patterns of vitamin D receptor in human prostate.

UV exposure and serum levels of vitamin D have been linked in several studies with prostate cancer risk. At the cellular level, the principal action of vitamin D is mediated though vitamin D receptors (VDR). Since prostate cancer is a disease strongly associated with age, we examined the presence of VDR in normal prostate from donors of various ages to determine if the VDR expression pattern changed with age. We also compared the VDR expression in the peripheral and central zones of the prostate to determine if the expression pattern varied by location. Immunohistochemical studies were performed on paraffin-embedded tissue from cases selected by the following age decades; 10-19, 20-29, 30-39, 40-49, 50-59, and 60-69. Both the central and peripheral zones were examined for VDR expression. The intensity of VDR expression in prostate was compared with expression in different types of human tissues. Mean VDR expression was lowest in the 10-19 years of age group. The intensity of the nuclear VDR was higher though the fifth decade, and then declined in cases of ages 60-70. When multiple sections of the same donor prostate were compared, VDR expression was greater in the peripheral zone compared to the central zone.

Efficacy of a synthetic lytic peptide in the treatment of prostate cancer.

In the last several years, significant effort has been applied to identifying novel agents with effectiveness against prostate cancer. These studies were designed to determine the efficacy of one of these novel compounds, D2A21, in the treatment of an animal model of prostate cancer. Using the Mat-Ly-Lu(MLL) line of the Dunning R-3327 rat prostate adenocarcinoma model, the optimal dose, schedule and route of administration of D2A21 were established. A study involving the G line was used to further support these findings. In addition, hemotoxylin and eosin stained tissue samples were examined to investigate the extent of inhibition of lung metastases in animals injected with MLL cells. When D2A21 was injected intraperitoneally or subcutaneously, MLL and G cell tumor growth was inhibited 50-72% as demonstrated by both tumor volumes and weights. The optimal dosage of 0.179 mg/injection was established and it was determined to be most efficacious when administered five times per week. At this concentration, D2A21 appears to have no significant toxicity. Additionally, D2A21 increased the survival rate from only 25% to 70-75% in animals that were challenged with a large number of tumor cells. The peptide D2A21 is able to significantly inhibit tumor growth in rat models of prostate cancer. In addition, it can inhibit metastases and decrease deaths resulting from metastases in these animals.

Urine based markers of urological malignancy.

PURPOSE: A number of urine based markers have been and are being investigated for the diagnosis and prognostication of urological conditions. A majority of these markers have been evaluated in urological neoplasms, particularly bladder cancer. The diagnosis of bladder cancer currently relies on identifying malignant cells in the urine and subsequently visualizing the tumor on cystoscopy. This diagnosis is further confirmed by transurethral resection or biopsy. While urine cytology is specific, it is not sensitive, especially for detecting low grade disease. This characteristic has prompted the search for more accurate markers of bladder cancer. In this review we critically examine the results of studies evaluating various markers for bladder cancer. MATERIALS AND METHODS: The published literature on urine based markers for all urological diseases, particularly bladder cancer, was identified using a MEDLINE search and critically analyzed. The sensitivity, specificity, positive and negative predictive values of the various markers were compared. The benefit of using combined markers rather than a single marker was also analyzed from published reports. RESULTS: Most published literature on urine based markers for urological malignancies involve such markers for diagnosing and prognosticating bladder cancer. Hence, we focused mainly on urine based markers in bladder cancer. Most markers appear to have an advantage over urine cytology in terms of sensitivity, especially for detecting low grade, superficial tumors. However, most markers tend to be less specific than cytology, yielding more false-positive results. This scenario is more common in patients with concurrent bladder inflammation or other benign bladder conditions. However, there is reason to be optimistic about several new markers that appear to provide better specificity. Few urine based markers have been identified and investigated in other urological tumors. CONCLUSIONS: Detecting bladder cancer using diagnostic markers still presents a challenge. A number of new markers are currently available that appear to be significantly more accurate than cytology. However, further studies involving a larger number of patients are required to determine their accuracy and widespread applicability for diagnosing bladder cancer. Urine based markers do not appear to have a significant role in the diagnosis or prognosis of other urological malignancies, such as prostate, kidney or testicular cancer.

Effects of vitamin D (calcitriol) on transitional cell carcinoma of the bladder in vitro and in vivo.

PURPOSE: Vitamin D (calcitriol) has significant antiproliferative effects on various tumor cells in vitro and in vivo. In the clinical situation a major impediment to systemic administration of calcitriol is the side effect of hypercalcemia. To test the potential usefulness of calcitriol for bladder cancer treatment, we studied the antiproliferative effect of vitamin D on 2 human bladder cancer cell lines, 253j and T-24, in vitro. We also examined the in vivo effects of calcitriol in an animal model of bladder cancer using intravesical administration to avoid the toxicity of systemic calcitriol therapy. MATERIALS AND METHODS: The presence of vitamin D receptors in normal and neoplastic human bladder tissue, and tumor cells T-24 and 253j was determined by immunoblot analysis. Tumor cell proliferation in the presence or absence of calcitriol was determined using a crystal violet assay. Calcitriol induced apoptosis was determined by morphology, polyadenosine diphosphate ribose polymerase cleavage and annexin V binding. In vivo studies were performed by weekly intravesical instillation of calcitriol in female Fischer 344 rats after induction of tumors by N-methyl nitrosourea. Calcitriol administration was started 3 weeks after tumor induction for 7 doses at weekly intervals. RESULTS: Normal and neoplastic human bladder tissue, and the cell lines expressed vitamin D receptors. In the 253j and T-24 cell lines proliferation was significantly inhibited by calcitriol. Progressive cleavage of full length polyadenosine diphosphate ribose polymerase was observed in calcitriol treated cells starting as early as 4 hours after exposure. Similar changes were not observed in the control cells treated with vehicle (ethanol) alone. After 24 hours of treatment with calcitriol 45.8% of 253j cells bound annexin compared to 16.5% of control cells (chi-square p <0.001). Of the control animals 66% developed bladder tumors and 55% of the animals treated with calcitriol early (3 weeks) after tumor induction developed bladder tumors. Almost all of the tumors that developed in the calcitriol group were unifocal, and only 20% were invasive compared to 50% of those in the control animals. CONCLUSIONS: These results demonstrate that calcitriol inhibits proliferation and induces apoptosis in human bladder tumor cells in vitro, and may have therapeutic potential in bladder cancer. In vivo studies using an N-methylnitrosourea induced model of bladder cancer demonstrate that early institution of intravesical calcitriol therapy after carcinogen exposure results in fewer tumors, which are also less likely to be multifocal, high grade or invasive. With our protocol a short course of intravesical calcitriol administration did not result in any significant toxicity.

In vitro and in vivo effects of vitamin D (calcitriol) administration on the normal neonatal and prepubertal prostate.

PURPOSE: Although many studies have investigated the role of calcitriol in the growth regulation of normal and cancerous prostates, little is known about its role in early prostatic development. The interactions between calcitriol and androgens, and their actions on the normal prostate have similarly been proposed but not evaluated. Previous studies in our laboratory have revealed that in utero administration of 1,25-dihydroxycholecalciferol or calcitriol can influence prostate growth and differentiation throughout the life of the animal. We further examined the influence of calcitriol on the normal prostate in vitro and in vivo by focusing on early stages of prostatic development. MATERIALS AND METHODS: The effects of calcitriol on the growth of the normal human neonatal prostatic epithelial cell line 267B-1 was determined in the presence and absence of dihydrotestosterone (DHT). We also examined the effect of calcitriol on the growth of maturing rat prostates in vivo. Before puberty 4 groups of rats 27 to 38 days old were treated with vehicle (controls) or calcitriol. When the rats reached adulthood at age 100 to 110 days a control group and a calcitriol group were sacrificed. The other 2 groups were given exogenous DHT for 5 days. For the animals to become adapted to DHT they were kept alive for 1 additional week and sacrificed at about age 120 days. RESULTS: In vitro studies demonstrated that 267B-1 cells possess vitamin D receptors and their growth was inhibited by calcitriol with an IC50 (concentration resulting in 50% cytotoxicity) of 30 microM. Proliferation of these neonatal prostate cells was also inhibited by calcitriol in the presence of DHT in vitro. Our studies indicate that, although calcitriol was administered at the apparently important prepubertal period, there was no difference in prostatic weights between the control and calcitriol treated rats. Exogenous administration of DHT decreased prostatic weight of control rats but in rats treated with 1,25-dihydroxycholecalciferol DHT did not have any significant effect on prostatic weight. No statistically significant differences were observed in seminal vesicle weights among the different groups of animals. Analysis of the nuclear matrix protein composition of the prostatic tissue showed differences in composition between the DHT, and calcitriol and DHT treated rat prostates. CONCLUSIONS: These studies indicate that calcitriol administered just before puberty does not significantly influence prostatic growth in the presence of endogenous or exogenous administered DHT, and has an inhibitory effect on neonatal prostate epithelial cell growth in vitro in the presence and absence of DHT. Treatment with calcitriol and DHT also results in differences in nuclear matrix protein composition. Prepubertal administration of calcitriol may inhibit the exogenous DHT action in decreasing epithelial growth and stimulating stromal proliferation in the rat prostate.

Clinical usefulness of the novel marker BLCA-4 for the detection of bladder cancer.

PURPOSE: Previous studies at our laboratory identified 6 bladder cancer specific nuclear matrix proteins termed BLCA-1 to 6. We recently developed an immunoassay that detects the bladder cancer specific nuclear matrix protein BLCA-4. We analyzed urine samples from patients with bladder cancer, those with spinal cord injury and normal volunteers to determine the BLCA-4 level in these 3 groups. MATERIALS AND METHODS: Urine samples obtained from 51 normal controls, and 54 patients with bladder cancer and 202 with spinal cord injury were tested for BLCA-4. We evaluated the association of BLCA-4 level with tumor grade and stage, urine cytology and bladder cancer history in the nonspinal cord injured population. Similarly we compared parameters associated with BLCA-4, such as spinal cord injury duration, catheterization, history of urinary tract infection, smoking and urine culture, in spinal cord injured patients. RESULTS: We established a normal cutoff point of 13 optical density units per microg. protein for the BLCA-4 assay. The BLCA-4 level was less than the cutoff in all 51 normal controls, while in 53 of the 55 urine samples (96.4%) of patients with bladder cancer and 38 of the 202 (19%) of spinal cord injured patients urinary BLCA-4 was greater than the cutoff. There was no correlation of any individual factors studied in these cases, including urinary tract infection and urinary BLCA-4. CONCLUSIONS: Elevated urinary BLCA-4 levels may accurately identify bladder cancer and distinguish these patients from normal individuals. There is no correlation of urinary BLCA-4 with a history of urinary tract infection, smoking, catheterization or cystitis considered independently. Urinary BLCA-4 determination appears to have high potential as a test for screening and monitoring bladder cancer in the general population and in groups at high risk for the disease, such as those with spinal cord injury.

Detection of bladder cancer using a novel nuclear matrix protein, BLCA-4.

We have identified previously six nuclear matrix proteins (NMPs) that are bladder cancer specific. In this study, we analyzed the expression of one of these proteins, BLCA-4, in bladder tumors and normal bladder tissue. We also examined the appearance of BLCA-4 in the urine as a biomarker for bladder cancer. BLCA-4 was isolated from nuclear matrix preparations of bladder tumors, and its peptide sequence was determined. The antibodies generated against the resulting BLCA-4 peptides were then used to detect its presence in immunoblots and in urine samples by immunoassay. We analyzed tissue samples of bladder tumor and normal donor bladders and urine obtained from 51 normal individuals and 54 patients with pathologically confirmed bladder cancer. The BLCA-4 peptide sequences do not resemble any known human protein sequences. On immunoblot analysis, BLCA-4 expression was detectable in tumor and normal tissues from patients with bladder cancer but not in any of the normal bladder tissue obtained from organ donors. Using a prospectively determined cutoff level of 13 A (absorbance) units/microg protein, all 51 normal individuals tested were negative for BLCA-4 expression, whereas 53 of 55 samples from patients with bladder cancer were positive. These results suggest that BLCA-4 is present throughout the bladder in both the tumor and morphologically normal areas in bladder cancer patients. BLCA-4 is a very sensitive (96.4%) and specific (100%) marker for bladder cancer. BLCA-4 is a bladder cancer-specific marker that can be detected using a urine-based assay and can be used in the diagnosis of bladder cancer.

Evaluation of the effect of spinal cord injury on serum PSA levels.

OBJECTIVES: Prostatic structure and secretory activity are thought to be influenced by autonomic innervation of the prostate. Prostatic denervation is especially likely in patients with spinal cord injury (SCI) at the level of the cauda equina or the conus medullaris, where the peripheral nerve supply to the prostate may be specifically damaged. This may result in changes in serum prostate-specific antigen (PSA) levels, either directly or indirectly. Therefore, we measured serum PSA levels and also studied the influence of factors such as age, catheterization, duration of SCI, urinary tract infection, and history of cystitis on serum PSA values in men with SCI. METHODS: Serum PSA levels were determined in 79 men with SCI (age older than 40 years) using banked sera by the Abbott MEIA PSA assay. Variables such as age, catheterization, duration of SCI, urine culture results, and history of cystitis were obtained from a review of patient records. Comparisons were made with a randomly selected, non-SCI control population of 501 men, 40 to 89 years old, who underwent serum PSA determination at our institution. Statistical comparisons were performed using the Mann-Whitney U test (nonparametric), since the populations were not normally distributed. Multivariate logistic regression analysis was used to assess the correlation between the various factors and the serum PSA levels in men with SCI. RESULTS: No statistically significant differences were found in the median serum PSA values between the SCI group and the non-SCI control population. The age-specific PSA values obtained in the SCI group were also comparable to those reported for the general population at large. Age (P <0.03) and the presence of a catheter (P <0.0002) were the only two factors that were correlated with higher serum PSA values in the SCI group by regression analysis. CONCLUSIONS: Men with SCI tended to have serum PSA value distributions that were similar to those of the general population. However, those in the SCI group who had indwelling catheters were more likely to have higher PSA values at baseline, as were older men with SCI.

Nuclear matrix proteins as cancer markers.

The transformation of normal cells to a malignant state has long been detected by light microscopy as visible changes in nuclear morphology. These changes include abnormal nuclear shape, increased nuclear to cytoplasmic ratio, and the presence of additional and abnormal nucleoli. Metaplasia,dysplasia, carcinoma-in-situ, and gross malignant tumors are diagnosed and graded pathologically by this traditional method. The resulting relative increase in DNA concentration within these cells produces a greater affinity for Hematoxilyn and Eosin staining, and thus, the characteristic blue color of cancerous tissues. As understanding of the cell structure expanded, the nuclear matrix emerged as an integral component of genetic processing and therefore, became an important cellular entity for study of malignant transformation. Also, several types of cancer have revealed discreet alterations in their respective nuclear matrices. One potential application of these nuclear matrix changes is development of detection and monitoring tests that would reveal the presence of abnormal cells. These tests could be utilized at a number of points in the disease process including prior to gross physical symptoms, and thereby significantly reduce patient morbidity and mortality. A second potential application of the nuclear matrix is to utilize it as a tissue specific protein targeting system to address narrowly directed therapeutic treatments, and thereby avoid the systemic side effects from broad-spectrum therapies like radiation. This paper addresses the role of the nuclear matrix in both normal cells and transformed cells, and highlights several research efforts that have advanced the ability to detect, track, and potentially treat neoplasms at the molecular level. J. Cell. Biochem. Suppl. 35:136-141, 2000.

Use of urine-based markers for detection and monitoring of bladder cancer.

Diagnosis and monitoring of bladder cancer present a difficult clinical problem. Urine cytology with confirmatory cystoscopy form the cornerstone of diagnosis at the present time. The subjectivity and low sensitivity of cytology led to the development of numerous tests as adjuncts to cystoscopy for the diagnosis and follow-up of bladder cancer patients. These tests usually are objective, quantitative (NMP-22, BTA TRAK, BLCA-4, telomerase activity, etc.), or qualitative (BTA Stat and FDP) and have higher sensitivity than cytology, but some have lower specificity. We review the different, new urine-based tests that were developed recently for the diagnosis and monitoring of patients with bladder cancer. The advantages and disadvantages of these tests are discussed, as well as their possible future role in the management of patients with bladder cancer.

Update on urine-based markers for bladder cancer. How sensitive and specific are the new noninvasive tests?

Urine cytology is still the most commonly used noninvasive test to diagnose bladder cancer. However, cytology's ability to detect low-grade bladder tumors is limited, and its results require interpretation by a pathologist, are not available immediately, and are subjective. Several noninvasive urine-based tests are now available for detection and follow-up of bladder cancer. At least two of these new tests (BTA stat and AuraTek FDP) can easily be performed in the office, and the results are available in about 10 minutes. When choosing a test, physicians should keep in mind that none of the currently available tests is 100% accurate. However, the new urine-based tests are more sensitive than urine cytology and hence more reliable in detecting low-grade bladder cancer. They are useful tools in patients with urinary symptoms or microscopic hematuria or as office-based adjuncts to diagnostic procedures. Some of the markers that are being developed could significantly improve and simplify workup, diagnosis, and follow-up, and they may allow for detection of disease at an earlier stage, thus improving the chances of curative therapy.

Effect of prenatal vitamin D (calcitriol) exposure on the growth and development of the prostate.

BACKGROUND: We previously found that in the absence of testosterone (T), calcitriol promotes proliferation of normal prostatic stroma, while in the presence of T, it has a differentiating effect on prostatic epithelium. The present study was conducted to determine the effect of calcitriol exposure in utero on the postnatal development of the normal prostate. METHODS: Pregnant rats were injected subcutaneously with either 1.25 microg of calcitriol or vehicle alone on alternate days till delivery. Calcitriol-exposed and control pups were sacrificed at age 25 days (prepuberty), 63 days (postpuberty), or 102 days (adults), and their prostates and seminal vesicles were harvested and weighed. RESULTS: Pups prenatally exposed to calcitriol and sacrificed before puberty (25 days) had a 35% greater mean prostatic weight than controls (0.0314 vs. 0.0422 g, P < 0.007), and calcitriol-exposed adult rats (102 days) had a 68% greater mean prostatic weight than controls (0.1365 vs. 0.2304 g, P < 0.005). No differences were observed in seminal vesicle weights, and in serum calcium and testosterone levels. A disproportionately high mortality rate from sudden death (71%) was observed at puberty in uncastrated male rats prenatally exposed to calcitriol. CONCLUSIONS: These findings suggest that high-dose calcitriol exposure in utero may uniquely influence subsequent prostatic growth. Nonandrogenic steroids such as calcitriol may also be involved in genetic imprinting of the prostate.

Differential effects of vitamin D on normal human prostate epithelial and stromal cells in primary culture.

OBJECTIVES: Because epidemiologic evidence has demonstrated that vitamin D may play a role in the etiology of prostate cancer, we tested the inhibitory effect of the biologically active form of vitamin D (1,25-D) on the cell proliferation of human prostate epithelial and stromal cells in a chemically defined situation in the presence and absence of dihydrotestosterone (DHT). We also tested the effect of 1,25-D in castrated rats in the presence and absence of flutamide, an androgen receptor blocker. METHODS: Prostate stromal and epithelial cells were isolated from freshly collected human prostatectomy specimens, and cell proliferation was measured with the MTT assay. Immunohistochemistry was performed to detect the presence of 1,25-D receptors, androgen receptors, smooth muscle actin, and E-cadherin. For in vivo analysis of 1,25-D, male Sprague-Dawley rats were castrated, then treated with either 1,25-D, 1,25-D with flutamide, or vehicle control. RESULTS: Incubation of primary cultures of prostate epithelial cells with 1,25-D at a concentration of 10(-8) M reduced cell proliferation by 40% of controls. The inhibition of growth by 1,25-D was maintained in the presence of DHT. Conversely, the effect of a similar dose of 1,25-D on stromal cell exposure was increased proliferation. In vivo, 1,25-D increased the prostatic weight of castrated rats that had serum testosterone levels below the detectable limit. The addition of flutamide did not alter this effect. CONCLUSIONS: These results confirm that vitamin D may be an effective antiproliferative agent of epithelial cells in prostate cancer therapy and support in vivo studies performed in the normal rat prostate.

Rat seminal-vesicle secretory protein SVS II binds DNA with a preference for the 5' regulatory region of secretory protein SVS IV gene: co-isolation with components of the nuclear matrix.

In rats, the ventral prostate and seminal vesicles produce distinct sets of proteins whose functions and tissue-specific regulation by androgens remain unclear. We have utilized the genes encoding the major secretory protein of seminal vesicles, SVS IV, and the C3 subunit of prostatein of the ventral prostate to study how the nuclear matrix might determine their tissue-specific gene expression. Nuclear matrix proteins were prepared from purified nuclei with DNase and 2 M NaCl, separated in SDS gels, and transferred onto membranes for DNA-binding (southwestern) and immunological (western) analyses. The 5' region of the SVS IV gene (SVS IV-7S) bound to a 45,000-kDa molecular-weight protein band in the nuclear matrix of seminal vesicles but not to that of ventral prostate, kidney, or liver. Sequencing revealed that this band was a seminal-vesicle secretory protein, SVS II, whose identity was confirmed with an anti-SVS II antiserum in western blots. Actin-like protein, similar in mobility to SVS II, was detected in seminal-vesicle and ventral prostate nuclear matrix, but not in seminal-vesicle fluid. Reducing agent (10 mM dithiothreitol) and acidic (pH 6.5) buffer did not eliminate SVS II, but isolation of nuclear matrices with ammonium sulfate, nucleases, and urea decreased SVS II immunoreactivity and removed actin-like protein. SVS II binding to SVS IV-7S DNA was greater than its binding to either a comparable fragment of the C3 gene or linearized pUC-19 plasmid, and it was not eliminated by a 100-fold competition. When seminal-vesicle fluid was mixed with rat liver, some SVS II co-isolated with the nuclear-matrix proteins, indicating that nonspecific interactions contribute to its association with the nucleoskeleton. However, these interactions may not represent the intracellular behavior of SVS II in seminal-vesicle epithelium. Sequence comparisons indicate significant homologies between SVS II and some other seminal proteins, including bovine caltrin, which, under the name seminalplasmin, is known to possess antimicrobial activity. Collectively, these data suggest that in addition to its known functions, SVS II may also bind extraneous DNA in seminal fluid. Additionally, SVS II may participate as a structural component in the organization of a tissue-specific seminal-vesicle nuclear matrix.

Vitamin D in the prevention and treatment of prostate cancer.

Current approaches to the management of prostate cancer include surgery, radiation therapy, or hormonal manipulation either individually or in combination. With an increase in understanding of the etiology and natural history of prostate cancer, the influence of dietary factors on the disease is becoming more evident. There have been a number of studies in this regard that have demonstrated a relationship between prostate cancer and numerous dietary constituents including vitamins. The fat-soluble vitamins A and D have both been found to affect the growth of prostate cancer in preclinical experiments. Of the two, vitamin D has been the focus of greater attention in recent years, and there are indications that it may be useful both in the prevention and treatment of prostate cancer. This article reviews the current literature in this area to determine if treatment with vitamin D would be a viable management alternative for the patient described in the case study.

Nuclear structural proteins as biomarkers of cancer.

The regulation of cell processes is integrally connected to cellular and extracellular structure. Studies over the past three decades have demonstrated the complex interactions of cell structure and function. The relationship of cellular structure and function has perhaps been most studied in the transformed cell. The hallmark of transformation is alterations in the shape of the cell and the nucleus. Many of the cellular alterations observed in the cancer process are structural, including changes in extracellular matrix-cytoskeletal interactions, cytoskeletal elements, as well as nuclear structure. This review focuses on the structural components of the nucleus, the nuclear matrix, and their role in the cancer process and the use of these structural components of the nucleus, the nuclear matrix, and their role in the cancer process and the use of these structural components as cancer specific biomarkers. J. Cell. Biochem. Suppls. 32/33:183-191, 1999.

Association of vitamin D receptors with the nuclear matrix of human and rat genitourinary tissues.

Calcitrol, 1,25 dihydroxyvitamin D3 (1,25-D3) has an important role in the antiproliferative and growth regulatory effects on normal and neoplastic cells (e.g. prostate cancer cells). 1,25-D3 binds to the vitamin D receptor (VDR), a member of the steroid receptor superfamily. Steroids, via intranuclear receptors, have been demonstrated to have high affinity binding to the nuclear matrix, the tissue specific scaffolding of the nucleus that is involved in the organization of DNA, replication and transcription. We hypothesized that the VDR interacts closely with the nuclear matrix in both human and rat tissues. In the studies described here, nuclear matrix proteins (NMP) were extracted from a number of rat and human tissues and immunoblot analysis performed using a rat anti-VDR antibody. The results from these studies reveal that the anti-VDR antibody detects six forms of the VDR in the NMP preparations: human testis demonstrated a protein of 57 and 52 kDa molecular weight compared with 57 and 37 kDa in the rat testis. Human prostate demonstrated proteins of 52 kDa compared to rat ventral (57 and 37 kDa) and dorsal prostate (52 and 26 kDa). Human and rat bladder NMP demonstrated a protein binding at 55 kDa and rat seminal vesicle NMP binding at 48 kDa. This is the first report of VDRs associated with the nuclear matrix. The varying molecular weight proteins reactive with the anti-VDR antibody within these tissues may represent different isoforms, proteolytic cleavage of a larger VDR or post-translational modification. The VDR-NMP interaction may be involved in the tissue specific actions of 1,25-D3 especially growth regulatory and antiproliferative effects.

The DNA-binding and tau2 transactivation domains of the rat glucocorticoid receptor constitute a nuclear matrix-targeting signal.

Using an ATP-depletion paradigm to augment glucocorticoid receptor (GR) binding to the nuclear matrix, we have identified a minimal segment of the receptor that constitutes a nuclear matrix targeting signal (NMTS). While previous studies implicated a role for the receptor's DNA-binding domain in nuclear matrix targeting, we show here that this domain of rat GR is necessary, but not sufficient, for matrix targeting. A minimal NMTS can be generated by linking the rat GR DNA-binding domain to either its tau2 transactivation domain in its natural context, or a heterologous transactivation domain derived from the Herpes simplex virus VP16 protein. The transactivation and nuclear matrix-targeting activities of tau2 are separable, as transactivation mutants were identified that either inhibited or had no apparent effect on matrix targeting of tau2. A functional interaction between the NMTS of rat GR and the RNA-binding nuclear matrix protein hnRNP U was revealed in cotransfection experiments in which hnRNP U overexpression was found to interfere with the transactivation activity of GR derivatives that possess nuclear matrix-binding capacity. We have therefore ascribed a novel function to a steroid hormone transactivation domain that could be an important component of the mechanism used by steroid hormone receptors to regulate genes in their native configuration within the nucleus.

Identification of nuclear matrix protein alterations associated with renal cell carcinoma.

PURPOSE: Neoplastic transformation, including renal cell carcinoma (RCC), is always accompanied by changes in nuclear morphology. Nuclear grading of RCC is based on characteristic alterations in nuclear shape, size, area and other morphologic parameters. The nuclear matrix, which forms the skeleton of the nucleus, determines nuclear morphology. Alterations in nuclear matrix protein (NMP) composition specific to tissue and cancer type have been described in a variety of human cancers. We conducted a study to analyze the nuclear matrix protein composition of renal cell carcinoma and compare it to that of normal renal tissue and renal cell carcinoma cells grown in culture. MATERIALS AND METHODS: We analyzed the nuclear matrix protein composition of RCC tumor tissue and that of normal kidney tissue obtained from seventeen patients undergoing radical nephrectomy for RCC. We also analyzed the NMP composition of two renal cancer cell lines (A-498 and 769-P). RESULTS: We were able to identify five different and unique NMPs which were present only in the human RCC tumor samples and were absent in all normal kidney tissue. One NMP was found specifically in the normal kidney tissue. All five RCC specific NMPs were also identified in the nuclear matrix of the two cell lines analyzed. CONCLUSIONS: Five nuclear matrix proteins specific and unique to RCC were identified. These NMPs are different from those previously identified in other tissues and neoplasms. The RCC specific NMPs identified in this study can potentially be used as diagnostic markers for renal cell carcinoma and for therapeutic tumor targeting.