Apolipoprotein-D: a novel cellular marker for HGPIN and prostate cancer.

BACKGROUND: High grade prostatic intraepithelial neoplasia (HGPIN) is a putative pre-malignant lesion of the prostate. While apolipoprotein-D (Apo-D), an androgen-regulated hydrophobic transporter protein, is expressed in prostate tumors, its expression in HGPIN is unknown. METHODS: Immunoreactivity for Apo-D and another androgen-regulated protein, prostate specific antigen (PSA), was investigated in 64 radical prostatectomy tissues by video image analysis. RESULTS: Eighty two percent of prostatectomy specimens demonstrated moderate to strong Apo-D immunoreactivity in areas of HGPIN. In comparison, weak Apo-D immunoreactivity was observed in non-malignant areas in only 24% of specimens. The median (range) percentage cellular area of HGPIN immunopositive for Apo-D (9.7%, 0-42.9), and the cellular concentration of Apo-D (MIOD 3.1, 0-13.3), were intermediate between that of normal (area 0%, 0-53.5%, MIOD 0, 0-12.6) and early stage prostate cancer tissues (area 29.2%, 0-90.8%, MIOD 6.7, 0-28.1). This increase in Apo-D expression from non-malignant, through HGPIN to prostate cancer was statistically significant (P < 0.001), and contrasted with the decrease observed in PSA staining between adjacent areas of normal glands, HGPIN, and cancer (P = 0.026). CONCLUSIONS: The presence of high levels of immunoreactive Apo-D in HGPIN and prostate cancer, but not in non-malignant epithelial cells, is consistent with HGPIN being an intermediate lesion in the transition to prostate cancer, and suggests that cellular Apo-D expression is a marker of malignant transformation of the prostate.

Simple, sensitive and accurate method for the quantification of prothrombin mRNA by using competitive PCR.

A method for the quantification of prothrombin (PT) mRNA species in hepatic tissues of rats was developed with the use of competitive PCR. To validate the quantification approach, sequential dilutions of total RNA from one of the samples were reverse transcribed. Their equivalent volumes were amplified together with a known amount of non-homologous competitor cDNA with identical nucleotide primers. The disparate sizes of target and competitor permitted the easy identification and quantification of bands in samples after densitometric analysis of ethidium bromide-stained agarose gels. Ratios of intensities of target and competitor bands were plotted against the initial amounts of total RNA species used, giving a linear relationship. The slope of this line was virtually identical with that obtained when the sample RNA was replaced with recombinant target cDNA, indicating that recombinant cDNA behaved in PCR identically with that made by reverse transcription and permitting the estimation of transcripts in reverse transcription reactions by using the recombinant counterpart of each as a standard. To avoid variation in the final results, the amount of competitor used in the assay was calculated separately from the equivalence point of the reverse-transcribed total RNA of one of the tissue samples; PCR was performed only for the minimum number of cycles required to detect products. A standard curve was made in each PCR run by amplifying differing amounts of recombinant cDNA species of PT or beta-actin together with a constant amount of its competitor. The numbers of transcripts in the tissues were then determined directly by PCR incorporating the same amount of respective competitor (as used in the standard curve) and comparing the ratios of products with the standard curve. Application of this method revealed that the median ratio of PT message to beta-actin message in hepatic tissues of 10 normal rats was 0.37, with a mean+/-S.D. of 0.37+/-0.07 (range 0.27-0.47). Although the method was developed for the quantification of PT transcripts in liver, it can easily be used for non-hepatic tissues as well. The technique is simple, quick and sensitive and requires only a very small amount of substrate.

Attitudes to evidence-based practice in urology: results of a survey.

BACKGROUND: The advantages of promoting evidence-based care through implementation of clinical guidelines are well established. Clinical practice guidelines have been developed for lower urinary tract symptoms (LUTS) and prostate cancer screening. Aspects of the delivery of care by urologists or specialist registrars relevant to the guidelines were assessed. METHODS: A questionnaire was distributed at the 1999 meeting of the Urological Society of Australasia, which was attended by 187 Australasian and 33 foreign delegates. Questions addressed access to resources for evidence-based medicine; perceived need; preferred sources of information; and then presented four clinical scenarios. These were: (i) treatment recommendations in early stage prostate cancer; (ii) the same scenario if the respondent was the patient; (iii) treatment recommendations after radical prostatectomy when there was a positive resection margin; and (iv) clinical investigations for mild to moderate LUTS. RESULTS: Of 220 possible responses, 132 were received, a response rate of 60%. Urologists overwhelmingly (100%) endorsed the need for access to evidence-based reviews, although 28% claimed such access was non-existent to poor. Clinical guidelines were the preferred source of evidence-based information. For early stage prostate cancer in a 55-year-old man, radical prostatectomy was recommended by 93.2% of respondents, but this dropped to 83% when the respondent was the patient (P < 0.05), and a wider range of treatments was recommended. Pelvic radiotherapy and hormone therapy were equally recommended for biochemical progression following radical prostatectomy where there was a positive surgical margin. Investigations for LUTS included serum prostate-specific antigen (PSA) testing (78.0%) and voided flow studies (77.3%). CONCLUSIONS: Urologists express a need for evidence-based practice resources, in particular clinical guidelines. Nevertheless their clinical approach is not necessarily consistent with existing guidelines, particularly for LUTS. An alteration in the recommendation when the respondent is the patient of interest and endorses the recommendation that patients with prostate cancer should be involved in treatment decisions.

CD40 is not detected on human prostate cancer cells by immunohistologic techniques.

OBJECTIVES: The CD40 antigen is expressed by antigen-presenting cells, many kinds of epithelium, and carcinomas. As signaling through CD40 modulates the differentiation state of CD40-expressing cells, we wanted to investigate whether benign or malignant prostate epithelium expressed CD40. METHODS: Twenty-two paraffin-embedded and 10 snap-frozen human prostate tissue samples were analyzed by immunohistologic methods, using the basal cell-specific markers, high molecular weight cytokeratin (HMWCK) and keratin-14 (K14), and the luminal cell marker, low molecular weight cytokeratin (LMWCK), together with CD40. Fresh prostate tissue was cultured in vitro and analyzed by immunocytofluorescence. RESULTS: The pattern of CD40 expression was continuous on basal epithelial cells of normal and hyperplastic prostate glands but discontinuous in glands that featured prostatic intraepithelial neoplasia. Coexpression of CD40 with the basal cell-specific cytokeratins, HMWCK and K14, was confirmed by double labeling. In contrast, glandular epithelial cells in prostate adenocarcinoma did not express CD40 or these cytokeratins. A luminal cell phenotype defined as CAM5.2-positive and HMWCK-negative K14-negative was identified among primary epithelial cells cultured in vitro. Most of the cultured cells (more than 99%) were also CD40-negative. CONCLUSIONS: Together, our results support the hypothesis that CD40 expression correlates with the basal cell phenotype, which is lost upon malignant transformation of the prostate. Hence, CD40 may be useful diagnostically to distinguish benign from malignant prostate lesions in biopsy material.

Factors predicting recovery of erections after radical prostatectomy.

PURPOSE: Because preservation of functioning penile erections is a major concern for many patients considering treatment for localized prostate cancer, we analyzed various factors determined before and after radical retropubic prostatectomy to identify those significantly associated with recovery of erectile function. MATERIALS AND METHODS: Our prospective database of patients undergoing pelvic lymphadenectomy and radical retropubic prostatectomy was used to determine factors predictive of erection recovery after radical prostatectomy. The study included 314 consecutive men with prostate cancer treated with radical retropubic prostatectomy between November 1993 and December 1996. Preoperative potency satisfactory for intercourse and degree of neurovascular bundle preservation during the operation were documented. RESULTS: Patient age, preoperative potency status and extent of neurovascular bundle preservation but not pathological stage were predictive of potency recovery after radical prostatectomy. At 3 years after the operation 76% of men younger than age 60 years with full erections preoperatively who had bilateral neurovascular bundle preservation would be expected to regain erections sufficient for intercourse. Compared to the younger men, those 60 to 65 years old were only 56% (95% confidence interval [CI] 37 to 84) and those older than 65 years were 47% (95% CI 30 to 73) as likely to recover potency. Patients with recently diminished erections were only 63% (95% CI 38 to 100) as likely to recover potency as men with full erections preoperatively, and those with partial erections were only 47% (95% CI 23 to 96) as likely to recover potency. Resection of 1 neurovascular bundle reduced the chance of recovery to 25% (95% CI 10 to 61) compared to preserving both nerves. CONCLUSIONS: Knowledge of preoperative erectile function and patient age before the operation and the degree of neurovascular bundle preservation afterward may aid in patient counseling regarding potency recovery after radical prostatectomy.

The prothrombin gene is expressed in the rat kidney: Implications for urolithiasis research.

There is considerable interest in determining the role of prothrombin fragments, especially urinary prothrombin fragment 1 (UPTF1), in the pathogenesis of calcium oxalate (CaOx) urinary calculi. This fragment is present in abundance in the matrix of CaOx crystals generated in human urine in vitro and has also been detected in human urinary stones containing calcium. More recently, prothrombin gene expression has been reported in the human kidney. However, studies examining the renal biosynthesis of prothrombin or perhaps only its fragments during experimental lithogenesis, and in consequence, the role of UPTF1 in stone formation, cannot be carried out in humans. The aim of this investigation therefore was to determine whether prothrombin gene expression is present in the rat kidney. Total RNA was isolated from the kidneys and livers of 12 rats. Using reverse transcriptase PCR, mRNAs corresponding to the thrombin and fragment 1 + 2 (F1+2) regions of prothrombin were analysed by agarose gel electrophoresis. The expression of glyceraldehyde 3-phosphate dehydrogenase was also examined to determine whether the quality of the tissue mRNAs was adequate for analyses. The amplified products were identified by sequence analysis. All kidneys displayed evidence of expression of the thrombin and F1+2 domains of the prothrombin gene. Furthermore, the sequences of these PCR-derived products from kidney were identical to those from liver. This suggests that the prothrombins secreted by these two organs are identical. The fact that prothrombin biosynthesis occurs in both the human and rat kidney presents an opportunity for using established rat models of stone disease to evaluate the influence of lithogenic conditions on prothrombin gene expression, and the potential role of UPTF1 in vivo.

Erectile dysfunction in the community: a prevalence study.

OBJECTIVE: To investigate the prevalence of erectile dysfunction (ED) in the South Australian community, and the influence of demographic and other risk factors. DESIGN: Survey by mailed questionnaire (based on the University of California, Los Angeles prostate cancer index) of a subset (men who agreed to participate) of a probability sample of the South Australian community who completed a multiuser interview survey. PARTICIPANTS AND SETTING: Men over the age of 40 in South Australia. MAIN OUTCOME MEASURES: Sexual desire, orgasm, ability to have an erection, adequacy (firmness) of erections for intercourse, frequency of erections when wanted, frequency of intercourse, nocturnal or morning erections, and history of prostate surgery; total sexual function score based on these. RESULTS: 612 men (86.7%) agreed to answer the sexual function survey; 427 (69.8%) returned questionnaires. ED was strongly correlated with age in all seven domains of sexual function. Erections inadequate for intercourse affected 3% of 40-49-year-olds, increasing to 64% of 70-79-year-olds. The frequency of intercourse considered normal for age by men 50-69 years was 1-6 times weekly; the disparity between this and reported frequency increased in men over 60 years, as did the difference between sexual desire and potency. A history of vigorous exercise was protective across all ages. High triglyceride levels, blood pressure medication and non-cancer surgery for prostate disease were independent predictors of poor sexual function at older ages. High cholesterol level was an independent predictor of impotence. CONCLUSIONS: We found similar or higher levels of ED than in comparable overseas studies. Disparity between potency and desire was greatest, and hence the age group in whom demand for treatment may be highest, in those 60 years and older. Cardiovascular risk factors were predictors of ED in these older men, suggesting that prevention may benefit sexual function. Non-cancer prostate surgery may be a greater contributor to ED than previously realised.

Prothrombin gene expression in rat kidneys provides an opportunity to examine its role in urinary stone pathogenesis.

Urinary form of prothrombin (PT) fragment 1 is the most abundant protein in calcium oxalate crystals generated in human urine. The protein has also been detected in human calcium-containing stones. In its purified form, the protein inhibits calcium oxalate crystal growth and, more importantly, aggregation in aqueous inorganic solutions and undiluted human urine. Recently, PT gene expression has been reported in human kidneys. However, access to human renal tissue for studies is limited, and it is not possible to easily manipulate PT biosynthesis in human subjects. The aim of this investigation, therefore, was to determine whether PT gene expression is present in rat kidneys. Samples of total RNA were isolated from the kidneys and livers (positive controls) of 12 male hooded Wistar rats. Using reverse transcription-PCR, mRNA corresponding to the thrombin and F1+2 regions of PT was analyzed by agarose gel electrophoresis. The expression of the "housekeeping" gene glyceraldehyde-3-phosphate dehydrogenase was also examined, to determine the availability of amplifiable substrate in each specimen. The amplified products were also sequenced, to determine their identities. All rat kidneys displayed evidence of expression of the thrombin and F1+2 domains of the PT gene. This similarity between human and rat kidneys allows the possibility of using established rat models of stone disease to evaluate therapeutic strategies to reduce stone formation.

Gene expression of prothrombin in the human kidney and its potential relevance to kidney stone disease.

OBJECTIVE: To determine whether urinary prothrombin fragment 1 (UPTF1), which shows considerable promise as a critical determinant of calcium oxalate (CaOx) stone formation, is manufactured by the human kidney. MATERIALS AND METHODS: Ribonucleic acid was isolated from eight kidneys, two spleens and one liver. Using reverse transcriptase-polymerase chain reaction, mRNA corresponding to the UPTF1 portion of prothrombin was analysed by agarose-gel electrophoresis and Southern blotting. RESULTS: Six kidney specimens showed clear evidence of prothrombin gene expression; expression in the kidney was less than that in the liver. CONCLUSION: This is the first demonstration of prothrombin gene expression within the human kidney, a finding that not only has implications for CaOx stone disease but also potentially for blood coagulation.

A preoperative nomogram for disease recurrence following radical prostatectomy for prostate cancer.

BACKGROUND: Few published studies have combined clinical prognostic factors into risk profiles that can be used to predict the likelihood of recurrence or metastatic progression in patients following treatment of prostate cancer. We developed a nomogram that allows prediction of disease recurrence through use of preoperative clinical factors for patients with clinically localized prostate cancer who are candidates for treatment with a radical prostatectomy. METHODS: By use of Cox proportional hazards regression analysis, we modeled the clinical data and disease follow-up for 983 men with clinically localized prostate cancer whom we intended to treat with a radical prostatectomy. Clinical data included pretreatment serum prostate-specific antigen levels, biopsy Gleason scores, and clinical stage. Treatment failure was recorded when there was clinical evidence of disease recurrence, a rising serum prostate-specific antigen level (two measurements of 0.4 ng/mL or greater and rising), or initiation of adjuvant therapy. Validation was performed on a separate sample of 168 men, also from our institution. RESULTS: Treatment failure (i.e., cancer recurrence) was noted in 196 of the 983 men, and the patients without failure had a median follow-up of 30 months (range, 1-146 months). The 5-year probability of freedom from failure for the cohort was 73% (95% confidence interval = 69%-76%). The predictions from the nomogram appeared accurate and discriminating, with a validation sample area under the receiver operating characteristic curve (i.e., comparison of the predicted probability with the actual outcome) of 0.79. CONCLUSIONS: A nomogram has been developed that can be used to predict the 5-year probability of treatment failure among men with clinically localized prostate cancer treated with radical prostatectomy.

Assessment of the biologic markers p53, Ki-67, and apoptotic index as predictive indicators of prostate carcinoma recurrence after surgery.

BACKGROUND: This study was designed to evaluate the potential of the molecular and cellular markers p53, Ki-67, and apoptotic index (AI) as adjuncts to the commonly available variables of tumor grade, clinical stage, and serum prostate specific antigen to predict prostate carcinoma recurrence after radical prostatectomy. METHODS: Representative punch biopsy specimens of prostate carcinoma from whole mount paraffin blocks were evaluated from 47 men who underwent radical prostatectomy. Two groups were defined: those without evidence of prostate carcinoma recurrence after 5 years of follow-up (N = 30) and those with carcinoma recurrence (N = 17). Gleason grade, clustered p53 immunostaining, Ki-67 immunostaining, and AI were determined by standard techniques. RESULTS: All variables tested were associated with disease recurrence by univariate analysis: AI (P = 0.005), clustered p53 immunostaining (P = 0.0070), and Ki-67 immunostaining (P = 0.0390). Using multivariate analyses that included each biomarker with routinely available features, only AI (P = 0.0234) and clustered p53 immunostaining (P = 0.0389) added independent prognostic information (Ki-67 immunostaining, P = 0.1285). In the final logistic regression model that included standard variables with AI and p53, only AI reached statistical significance (P = 0.0332). CONCLUSIONS: The continued assessment of additional biomarkers for prostate carcinoma recurrence is important to identify better those patients who may be candidates for early adjuvant therapy and also to further our understanding of the neoplastic potential of a particular malignancy.

Primary human prostate cancer cells harboring p53 mutations are clonally expanded in metastases.

Recent studies suggest a role for p53 in prostate cancer progression. Although p53 mutations in primary prostate cancer tissues are relatively infrequent, they occur at significant levels in metastatic disease. Here we describe a novel approach to the molecular analysis of p53 in paired specimens of primary and metastatic prostate cancer that results in quantitative estimates of the extent of clonal expansion. In 20 pairs with 1 or both specimens p53 immunopositive and in 6 pairs with both specimens immunonegative, the frequency of mutations was estimated by microdissection of the cancer from fixed and sectioned tissues, isolation of the DNA followed by PCR amplification of p53 genomic fragments, and cloning of the PCR products into plasmid vectors. At least 90 clones/tissue specimen were screened for mutations by single-strand conformational polymorphism analysis. DNA from abnormally migrating single-strand conformational polymorphism samples was sequenced to confirm mutations. Missense mutations in exon 5, 7, or 8 were detected in 9 of 20 immunopositive pairs and in 1 of 6 immunonegative pairs. A marked heterogeneity of mutations in primary prostate cancer was apparent. The frequency of p53 mutations was greater in the metastases than in the primary tumors. In three immunopositive pairs, the same p53 mutation was demonstrated at a low frequency in the primary tumor but was demonstrated at a greater frequency in the metastasis, indicating relatively limited clonal expansion of cells harboring specific p53 mutations in the primary tumor, yet significant clonal growth at metastatic sites as determined by this novel method.

Evaluation of a nomogram used to predict the pathologic stage of clinically localized prostate carcinoma.

BACKGROUND: A Nomogram based on pretreatment prostate specific antigen (PSA) level, tumor grade, and clinical stage has recently been developed and distributed to physicians. It was distributed to aid physicians in making treatment recommendations by predicting the probability of the final pathologic stage of clinically localized prostate carcinoma. The Nomogram was based on data for one patient population, and the validity of its application in general urologic practices had not yet been evaluated. METHODS: In the current study, the authors tested the performance of the Nomogram against data from their series of 697 men who underwent radical prostatectomy during the PSA era. Predictions made with the Nomogram were applied to the authors' data set, and the predictions were compared with actual outcomes of the authors' patients. A localized least-squares regression smoothing technique was used to determine whether the Nomogram was calibrated accurately for the authors' data and whether it discriminated across a full spectrum of patient characteristics. RESULTS: Many of the predicted probabilities of the Nomogram were accurate, but some were suboptimal when applied to the authors' data set. Although the Nomogram did discriminate quite well between organ-confined and nonconfined cancer, it had difficulty predicting high probabilities of seminal vesicle invasion and lymph node metastasis, which are the pathologic features with the most profound impact on prognosis. CONCLUSIONS: The Nomogram predicted organ-confined disease accurately. However, because not all of its predictions were completely calibrated when applied to the authors' data set, the authors conclude that the Nomogram may not be totally applicable to general urologic practice until further validation and possible modifications are performed.

Human cytomegalovirus is not implicated in benign prostatic hyperplasia: a study using immunohistochemistry and the polymerase chain reaction.

PURPOSE: To evaluate whether human cytomegalovirus (CMV) may play a role in the pathogenesis of benign prostatic hyperplasia. MATERIALS AND METHODS: We used immunohistochemistry and the polymerase chain reaction (PCR) to evaluate the presence of CMV in normal prostatic tissue (n = 12) and BPH tissue (n = 21). RESULTS: Immunostaining for CMV in prostatic tissue was negative; however, staining was evident in mononuclear cells seen in the tissues of both groups. With nested PCR, CMV genomic material was demonstrated in 1 tissue specimen from each group (p > 0.1). CONCLUSIONS: These data suggest that CMV is unlikely to be a significant factor in the pathogenesis of BPH.

Reduced levels of transforming growth factor beta receptor type II in human prostate cancer: an immunohistochemical study.

In previous studies we demonstrated that the growth of human prostatic adenocarcinoma is associated with aberrant accumulation of transforming growth factor (TGF) beta1, a growth factor that has been shown to be a potent inhibitor of epithelial cell proliferation. We investigated the expression of TGF-beta receptor II (TGFbetaR-II) in benign prostate tissue and in prostate cancer using standard immunohistochemical techniques. Quantitation of immunopositivity for TGFbetaR-II was assessed on a visual analogue scale ranging from 0 (absence of staining) to 4+ (intensely positive staining). All of the benign glandular epithelia stained intensely, either 3+ or 4+, representative of the ubiquitous nature of TGFbetaR-II in normal tissue. Overall, staining was reduced in prostate cancer sections, and there was progressively diminished staining as the histological grade of the cancer increased (P < 0.01, Kruskal-Wallis test). This immunohistochemical study indicates that a decline in the levels of TGFbetaR-II is correlated with advancing histological aggressiveness of the cancer and suggests that aberrant TGFbetaR-II function may play a role in human prostate carcinogenesis.

Further evidence linking urolithiasis and blood coagulation: urinary prothrombin fragment 1 is present in stone matrix.

The fact that organic material is always present and distributed throughout each renal calculus suggests that it may play a role in stone formation. The organic matrix of calcium oxalate (CaOx) crystals freshly generated in urine in vitro contains urinary prothrombin fragment 1 (UPTF1) as the principal protein. In this initial study, matrix was extracted from 12 renal calculi and evaluated for the presence of UPTF1 using Western blotting. UPTF1 was present in all eight stones whose principal component was CaOx, and in one of two stones which consisted mainly of calcium phosphate (CaP). UPTF1 was absent from the two struvite calculi examined. The relationship between CaP and UPTF1 was explored further. Matrix harvested from CaP crystals freshly generated in urine in vitro was also shown to contain UPTF1 as its principal component. Our inability to detect UPTF1 in one mixed CaOx/CaP stone may be related to our methods of matrix retrieval, while its absence from two struvite stones argues against it being present in the other stones merely as a consequence of passive inclusion. This absence may be related to the alkaline environment typical of struvite stone growth. The finding that UPTF1 is present in some renal stones provides the first direct evidence that links blood coagulation proteins with urolithiasis.

Clustered p53 immunostaining: a novel pattern associated with prostate cancer progression.

Abnormal p53 protein accumulation is typically defined as present when greater than 5 or 10% of cancer cells stain positively. We present a novel approach whereby immunopositivity is defined when 15 or more cells within a 300 x 400-micrometer(2) field exhibit p53 protein accumulation; a feature that we have called "clustered" staining. We assessed p53 immunostaining of moderately differentiated, clinically localized prostate cancers derived from two patient groups: those without cancer recurrence 5 years after radical prostatectomy, and those in whom cancer had recurred following radical prostatectomy. Clustered p53 immunopositivity was present in 10 (63%) of 16 patients in the recurrent group and in only 7 (21%) of 33 in the nonrecurrent group. Clustered p53 staining was clearly associated with cancer recurrence (P < 0.01). This refinement of a commonly used assay may help define the biological aggressiveness of a cancer.

The urinary F1 activation peptide of human prothrombin is a potent inhibitor of calcium oxalate crystallization in undiluted human urine in vitro.

1. The urinary F1 activation peptide of prothrombin is the predominant protein incorporated into calcium oxalate crystals precipitated from human urine. The aim of this study was to examine the effect of pure urinary prothrombin F1 on calcium oxalate crystallization in human urine. 2. Urinary prothrombin F1 was purified from demineralized calcium oxalate crystals precipitated from human urine, and its effects on calcium oxalate crystallization induced by addition of an oxalate load were tested in undiluted, ultrafiltered urine from healthy men, at final concentrations of 0 to 10 mg/l. 3. Urinary prothrombin F1 did not affect the amount of oxalate required to induce crystallization, but the volume of material deposited increased in proportion to increasing concentrations of urinary prothrombin F1. However, the mean particle size decreased in reverse order: this was confirmed by scanning electron microscopy, which showed it to be the result of a reduction in crystal aggregation rather than in the size of individual crystals. Analysis of 14C-oxalate data revealed a dose-dependent decrease in calcium oxalate deposition with an increase in urinary prothrombin F1 concentration, indicating that the increase in particle volume recorded by the Coulter Counter resulted from inclusion of urinary prothrombin F1 into the crystalline architecture, rather than increased deposition of calcium oxalate. 4. It was concluded that urinary prothrombin F1 may be an important macromolecular determinant of stone formation.

Association of p53 mutations with metastatic prostate cancer.

In prostate cancer, mutation of the p53 tumor suppressor gene has been associated with locally advanced disease and hormone-resistant disease that is predominantly localized to bone. However, little is known regarding the status of the p53 gene in metastatic prostate cancer that has not been treated with hormonal manipulation. We evaluated formalin-fixed, paraffin-embedded malignant tissues from 86 patients with various stages of prostate cancer, including pathologically confined, locally advanced, and metastatic disease, to detect abnormal p53 nuclear protein accumulation using immunohistochemistry. No abnormal p53 immunostaining was detected in 18 patients with prostate cancer confined to the gland. Two tumors from 21 patients with locally advanced disease (extracapsular extension and/or seminal vesicle invasion) had abnormal nuclear p53 accumulation, and a mutation in exon 7 of the p53 gene was detected in tumor DNA from one patient using single-strand conformation polymorphism-direct sequencing analysis. Of the remaining 47 patients studied in whom tissues from the prostate gland and a metastatic site (44 lymph node, 2 bone, and 1 lung) were available, only 3 had received hormonal therapy prior to obtaining metastatic tissue. In four patients both primary and metastatic tumors demonstrated accumulation of p53 protein, whereas seven additional patients exhibited p53 accumulation only at the metastatic site. In three patients the metastatic tumors harbored missense single-base substitutions in exon 5, as detected using single-strand conformation polymorphism-direct sequencing. These results indicate that p53 abnormalities are associated with lymph node metastases derived from prostate cancer patients that had not undergone hormonal therapy.

Blood coagulation proteins and urolithiasis are linked: crystal matrix protein is the F1 activation peptide of human prothrombin.

OBJECTIVES: To determine the relationship between prothrombin and crystal matrix protein (CMP). CMP is the predominant protein found in the organic matrix of calcium oxalate (CaOx) crystals generated from human urine and is a 31 kDa glycoprotein, whose N-terminal amino acid sequence shares homology with human prothrombin. MATERIALS AND METHODS: CaOx crystallization was induced in ultrafiltered (UF) human urine containing either plasma or serum derived from the same healthy donor, by the addition of sodium oxalate. The crystals were demineralized and the resulting protein extracts analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, using antibodies raised against human prothrombin and the C-terminus of prothrombin fragment 1 + 2 (F1 + 2). RESULTS: Prothrombin was detected in extracts of crystals precipitated from the UF urine in the presence of plasma, while CMP was completely absent. Crystals precipitated from UF urine supplemented with serum contained relatively large amounts of F1 + 2 and a protein with the same electrophoretic mobility as CMP. Analysis of a standard preparation of F1 + 2 which also contained prothrombin fragment 1 (F1) as a minor contaminant, showed a protein with electrophoretic and staining properties comparable to CMP. CONCLUSION: CMP is a urinary form of F1, a degradation product of prothrombin possessing the domain rich in gamma-carboxyglutamic acid, which may have undergone some molecular modification either before or after its release into the urine.