[Cost-effectiveness analysis of octreotide long acting release and lanreotide slow release for the treatment of postoperative patients with active acromegaly in China].

Objective: To evaluate the cost-effectiveness of octreotide long acting release (LAR) vs lanreotide slow release (SR) for the treatment of postoperative acromegalic patients with elevated levels of growth hormone (GH) and insulin-like growth factor 1 (IGF-1) in China. Methods: A decision tree model was constructed and the treatment impact was projected for one year in Chinese setting. The clinical efficacy measure used was the percentage of patients achieving normalization (control) of either IGF-1 or GH levels. Efficacy of octreotide LAR and lanreotide SR, incidence of comorbidities, impact of acromegaly on health-related quality of life, and drug-related side effects data were obtained from literature. The cost of medication was collected through a chart review from five hospitals in five cities of China. Clinical experts from these hospitals were requested to complete a questionnaire to document the utilization of medical resources, costs of comorbidities, side effects as well as cost of administration. One-way sensitivity analysis was performed to evaluate the robustness of the results. Results: Compared to lanreotied SR group, the percentage of patients achieving normalization of IGF-1 and GH levels of octreotide LAR group were 10% and 9% higher, respectively. When either IGF-1 or GH control were used as the efficacy measure, patients in the octreotide LAR group exhibit less comorbidities and need less continued treatment with a second operation and radiotherapy than those in lanreotide SR group. When IGF-1 was used as efficacy measure, octreotide LAR not only achieved better efficacy but resulted in overall cost-saving, with a total cost savings of ï¿¥ 3 792 per patient for one year, which demonstrated that octreotide LAR was a dominant cost-saving strategy. When GH control was used as the efficacy measure, octreotide LAR achieved a better overall clinical efficacy with a slightly higher total costs (ï¿¥ 4 121 higher per patient per year). Sensitivity analysis didn't change the conclusion that octreotide LAR remains dominant over lanreotide SR, indicating the robustness of this model. Conclusion: Octreotide LAR achieved better overall biochemical control compared with lanreotide SR which result in less comorbidity rate, second operation and radiotherapy as well as related costs.

Modeling the cost-effectiveness of ilaprazole versus omeprazole for the treatment of newly diagnosed duodenal ulcer patients in China.

OBJECTIVES: To evaluate the cost-effectiveness of 10 mg ilaprazole once-daily vs 20 mg omeprazole once-daily to treat newly-diagnosed duodenal ulcer patients in China. METHODS: A decision tree model was constructed and the treatment impact was projected up to 1 year. The CYP2C19 polymorphism distribution in the Chinese population, the respective cure rates in the CYP2C19 genotype sub-groups, the impact of Duodenal Ulcer (DU) on utility value and drug-related side-effect data were obtained from the literature. The total costs of medications were calculated to estimate the treatment costs based on current drug retail prices in China. Expert surveys were conducted when published data were not available. Probabilistic sensitivity analysis was performed to gauge the robustness of the results. RESULTS: Ilaprazole, when compared with omeprazole, achieved a better overall clinical efficacy. For the overall population, ilaprazole achieved an incremental cost effectiveness ratio (ICER) of ¥132 056 per QALY gained. This is less than the WHO recommended threshold of 3-times the average GDP per capita in China (2014). Furthermore, sub-group analysis showed that ilaprazole is cost-effective in every province in CYP2C19 hetEM patients and in the most developed provinces in CYP2C19 homEM patients. Probabilistic sensitivity analysis suggests that the results are robust with 97% probability that ilaprozole is considered cost-effective when a threshold of 3-times China's average GDP per capita is considered. LIMITATION: This study didn't have the data of ilaprazole combined with Hp eradication therapy. Caution should be taken when extrapolating these findings to DU patients with an Hp eradication therapy. CONCLUSIONS: The cost-effectiveness analysis results demonstrated that ilaprazole would be considered a cost-effective therapy, compared with omeprazole, in Chinese DU patients based on the efficacy projections in various CYP2C19 polymorphism types.

Histopathological characteristics of a novel knock-in mouse prostate cancer model.

Prostate cancer is relatively unique to man. There is no naturally occurring prostate cancer in the mouse. Pre-clinical studies involve the establishment of a genetically engineered mouse prostate cancer model with features close to those of the human situation. A new knock-in mouse adenocarcinoma prostate (KIMAP) model was established, which showed close-to-human kinetics of tumor development. In order to determine if the similar kinetics is associated with heterogeneous tumor architecture similar to the human situation, we utilized a new mouse histological grading system (Gleason analogous grading system) similar to the Gleason human grading system and flow cytometry DNA analysis to measure and compare the adenocarcinoma of the KIMAP model with human prostate cancer. Sixty KIMAP prostate cancer samples from 60 mice were measured and compared with human prostate cancer. Flow cytometry DNA analysis was performed on malignant prostate tissues obtained from KIMAP models. Mice with prostate cancer from KIMAP models showed a 53.3% compound histological score rate, which was close to the human clinical average (50%) and showed a significant correlation with age (P = 0.001). Flow cytometry analyses demonstrated that most KIMAP tumor tissues were diploid, analogous to the human situation. The similarities of the KIMAP mouse model with tumors of the human prostate suggest the use of this experimental model to complement studies of human prostate cancer.

Histopathological characteristics of a novel knock-in mouse prostate cancer model

Prostate cancer is relatively unique to man. There is no naturally occurring prostate cancer in the mouse. Pre-clinical studies involve the establishment of a genetically engineered mouse prostate cancer model with features close to those of the human situation. A new knock-in mouse adenocarcinoma prostate (KIMAP) model was established, which showed close-to-human kinetics of tumor development. In order to determine if the similar kinetics is associated with heterogeneous tumor architecture similar to the human situation, we utilized a new mouse histological grading system (Gleason analogous grading system) similar to the Gleason human grading system and flow cytometry DNA analysis to measure and compare the adenocarcinoma of the KIMAP model with human prostate cancer. Sixty KIMAP prostate cancer samples from 60 mice were measured and compared with human prostate cancer. Flow cytometry DNA analysis was performed on malignant prostate tissues obtained from KIMAP models. Mice with prostate cancer from KIMAP models showed a 53.3 percent compound histological score rate, which was close to the human clinical average (50 percent) and showed a significant correlation with age (P = 0.001). Flow cytometry analyses demonstrated that most KIMAP tumor tissues were diploid, analogous to the human situation. The similarities of the KIMAP mouse model with tumors of the human prostate suggest the use of this experimental model to complement studies of human prostate cancer.

Application of Gleason analogous grading system and flow cytometry DNA analysis in a novel knock-in mouse prostate cancer model.

OBJECTIVE: A new knock-in mouse adenocarcinoma prostate model (KIMAP) was established, which showed a close to human kinetics of tumour development. This study used a new mouse histological grading system similar to the human Gleason grading system and flow cytometry DNA analysis to measure and compare the new KIMAP model with human CaP and transgenic mouse adenocarcinoma prostate (TGMAP) model. METHODS: According to heterogeneity of the clinical standard for prostate cancer diagnosis, a close to human mouse standard for histological grading and scoring system, Gleason analogous grading system, was established in this study. Sixty KIMAP and 48 TGMAP prostate cancer samples were measured and compared with human CaP. Flow cytometry DNA analysis was performed on malignant prostate tissues obtained from both TGMAP and KIMAP models. RESULTS: Mice with CaP from KIMAP (n = 60) and TGMAP (n = 48) models showed a different distribution of histological scores (p = 0.000). KIMAP mice showed higher percentage (53.3%) of compound histological score rate than TGMAP (25%), but closer to the human clinical average (50%), which showed significant correlation with age (p = 0.001), while TGMAP mice showed unbalanced and random score distribution in all age groups. Flow cytometry analyses showed that most tumour tissues in KIMAP were diploid, analogous to the human condition, while all the TGMAP mice showed aneuploid tumours. CONCLUSIONS: Results of this study further show that KIMAP, a new generation of murine prostate cancer model, could be used as a supplementary model in addition to the currently widely used transgenic models.

Expression study of three secretory proteins (prostatic secretory protein of 94 amino acids, probasin, and seminal vesicle secretion II) in dysplastic and neoplastic rat prostates.

BACKGROUND: Prostatic secretory protein of 94 amino acids (PSP94), probasin, and seminal vesicle secretion II (SVSII) are the three major proteins secreted by the lateral lobe of the rat prostate gland. Among these proteins, rodent PSP94 but not probasin and SVSII has a human homologue and it is also a major secretory protein of the human prostate, in addition to prostatic acid phosphatase and prostate-specific antigen. METHODS: In this study, we examined and compared the mRNA expression of these three secretory markers in three rat models of prostate cancer including the sex steroid-induced dysplasia (prostatic intraepithelial neoplasia or PIN) in Noble (Nb) rat model, an androgen-independent Nb rat prostatic tumor (AIT) and Dunning rat prostatic adenocarcinomas (both androgen-dependent and -independent) by in situ hybridization (ISH), reverse transcriptase-polymerase chain reaction (RT-PCR), and immunohistochemistry. RESULTS: The transcripts for the three markers were highly expressed in the secretory epithelium of normal lateral prostate (LP). Their hybridization signals became reduced in the epithelial cells in the low-grade PINs and significantly weakened or lost in the high-grade PINs induced in the LP. Interestingly, we observed that some dysplastic cells located at the basal compartment of the PIN lesions, and nests of outpouching epithelial cells in the vicinity of PINs, expressed positive hybridization signals of three markers. In the adenocarcinoma, signals of probasin but not PSP94 and SVSII were detected. No hybridization signals were detected in both Dunning and AIT tumors. By RT-PCR, transcripts for these proteins were still detected but significantly reduced in the Dunning tumors, whereas in the AIT tumor, only SVSII transcripts were detected. Immunohistochemistry of PSP94 also showed a reduced staining in the PIN lesions, but no immunoreactivity was seen in the rat prostatic tumors. CONCLUSIONS: The mRNA expression of the three prostatic secretory markers were decreased in the hormone-induced PINs and in two rat prostatic tumors, indicating that the androgen-regulated secretory differentiation was impaired during the development of the premalignant lesion and further reduced in advanced tumors. The abnormal expression pattern of these secretory markers and androgen receptor (AR) in the basal compartment of the PIN lesions suggests that there is a population of cell types with secretory phenotype appearing in the basal cell layer during the early malignant transformation of the prostatic epithelium.

Prostate targeting: PSP94 gene promoter/enhancer region directed prostate tissue-specific expression in a transgenic mouse prostate cancer model.

To date, only a few prostate-specific vector genes have been tested for prostate targeting in gene therapy of prostate cancer (CaP). Current clinical trials of gene therapy of CaP utilize the only two available vector genes with a combination of a rat probasin promoter and a human PSA promoter sequence in an adenovirus vector to target CaP. There is an urgent need to establish additional vector gene systems to sustain and propagate the current research. Since PSP94 (prostate secretory protein of 94 amino acids) is one of the three most abundant proteins secreted from the human prostate and is generally considered to be prostate tissue-specific in both human and rodents, we performed a transgenic experiment to assess the promoter/enhancer region of PSP94 gene-directed prostate targeting. Firstly, a series of progressive deletion mutants of a 3.84 kb PSP94 gene promoter/enhancer region (including parts of the intron 1 sequence) linked with a reporter LacZ gene was constructed and assessed in vitro in cell culture. Next, transgenic mice were generated with two transgene constructs using the SV40 early region (Tag oncogene) as a selection marker. PSP94 gene promoter/enhancer region-directed SV40 Tag expression specifically in the mouse was demonstrated in three breeding lines (A, B, C, n = 374) by immunohistochemistry staining of Tag expression. Specific targeting to the prostate in the PSP94 gene-directed transgenic CaP model was characterized histologically by correlation of SV40 Tag-induced tumorigenesis (tumor grading) with puberty and age (10-32 weeks). Prostatic hyperplasia was observed as early as 10 weeks of age, with subsequent emergence of prostatic intraepithelial neoplasia (PIN) and eventually high grade carcinoma in the prostate. The PSP94 transgenic mouse CaP model was further characterized by its tumor progression and metastatic tendency at 20 weeks of age and also by its responsiveness and refractoriness to androgen manipulation. This study indicates that the PSP94 gene promoter/enhancer has the potential for prostate specific targeting and may ultimately be of use in gene therapy of CaP.

Rodent PSP94 gene expression is more specific to the dorsolateral prostate and less sensitive to androgen ablation than probasin.

To date, the rodent ventral prostate (VP) has been the focus of many studies on androgen action, less attention has been directed to the lateral prostate (LP) and the dorsal prostate (DP). The rodent VP has no clear homologous counterpart in the human prostate. The rodent LP and DP is the only prostate lobe comparable to the peripheral zone of the human prostate, where hormone-induced prostate cancer mainly occurs. To explore its utility for prostate targeting, we have studied the gene expression of PSP94 with rat probasin (rPB), a gene commonly used for prostate targeting in prostate cancer research and a gene typically responsive to androgen regulation. Firstly, we demonstrated PSP94 gene transcription being more specific to the LP and DP lobes than rPB, where rPB RNA was detected in the LP and DP and other lobes at different levels. Secondly, we found that PSP94 gene transcription decreased relatively slowly in response to androgen deprivation but recovered rapidly in response to testosterone replacement after complete ablation of PSP94 transcription. In the VP, gene transcripts of rPB were specifically responsive to androgen deprivation; however, they responded relatively slowly in the LP and DP. RNase protection experiments indicated that the slow response was not due to abnormal persistence of PSP94 messenger RNA specifically in the DP and LP lobes in comparison with rPB. Thirdly, Western blot analysis revealed that both PSP94 and rPB expression is specific to the LP and DP at the protein level, exhibiting slow responses to testosterone replacement after castration. We conclude that PSP94 gene expression at the transcriptional level is more specific to the LP and DP than rPB and thus less sensitive to androgen ablation. This may have clinical implications for strategies to target the prostate in cancer therapy.

A comparative study of hormonal regulation of three secretory proteins (prostatic secretory protein-PSP94, probasin, and seminal vesicle secretion II) in rat lateral prostate.

The rat dorsolateral prostate secretes several major known proteins, although their physiological and reproductive functions are largely undefined. In the present study we examined and compared the in vivo hormonal regulation of the messenger RNA (mRNA) expression of three major secretory proteins, including prostatic secretory protein of 94 amino acids (PSP94 or beta-microseminoprotein), probasin, and seminal vesicle secretion II (SVSII), in long-term castrated lateral prostates (LP) by in situ hybridization and semiquantitative RT-PCR. The protein levels of PSP94 in the castrated LPs were also examined by Western blotting. PSP94 is a small protein newly isolated from the rat prostate gland and demonstrates highly specific expression in the LP. The results of in situ hybridization showed that PSP94, probasin, and SVSII were highly expressed in the intact LP. The hybridization signals of probasin and PSP94 disappeared in the 60-day postcastrated LPs, whereas the signals of SVSII dropped sharply in the 14-day postcastrated LPs. Similar patterns of decreasing mRNA levels of the three proteins in the castrated LPs were observed by RT-PCR analysis. Their mRNA transcripts were restored to normal levels after replacement with testosterone. The results indicate that these secretory proteins are all under androgen regulation in the rat LP. Interestingly, we also observed that their degrees of sensitivity or responsiveness to androgen withdrawal are different. Their mRNA levels dropped in response to duration of castration in the following decreasing order: SVSII, PSP94, and probasin. Besides androgen [dihydrotestosterone (DHT)], we also examined the effects of glucocorticoid [dexamethasone (DEX)], progestin [medroxyprogesterone acetate (MPA)], and zinc on their gene expressions in castrated LPs. We observed that the mRNA transcripts of both PSP94 and probasin were increased after treatments with DHT, DEX, and MPA, suggesting that these two proteins could also be regulated by glucocorticoid and progestin. In contrast with probasin, PSP94 and SVSII were not induced by ZnSO4 treatment. On the other hand, SVSII expression was only increased significantly by DHT and moderately by MPA, but not by DEX, suggesting that SVSII is under strict control by androgen.

PSP94 expression after androgen deprivation therapy: a comparative study with prostate specific antigen in benign prostate and prostate cancer.

PURPOSE: To examine the clinical use of PSP94 (prostate secretory protein of 94 amino acids) as an androgen independent marker, we conducted a comparative study of prostate samples including benign tissue and cancers which did and did not have androgen deprivation. MATERIALS AND METHODS: Among 163 radical prostatectomy cases 75 had androgen deprivation before operation, while surgery was performed in the remainder without prior hormone treatment. Considering the pathological up grading following hormone therapy, contiguous sections from radical prostatectomy samples were stained for PSP94 and prostate specific antigen (PSA) by immunohistochemistry, and equivalent tumor foci were evaluated by assessing the intensity and extent of the staining. RESULTS: In untreated benign prostate tissue PSP94 and PSA staining was positive and identical in all sections in the no pretreatment group. However, PSP94 expression in the androgen deprivation group was significantly higher than PSA in intensity (p = 0.0005) and extent (p = 0.034). In untreated cancer cases PSP94 intensity and extent demonstrated strong inverse association with Gleason grade (p <0.0001). In contrast, PSA expression was high in every grade, resulting in no statistical association with tumor grade. In the androgen deprivation group PSA staining was decreased in every grade compared to the no pretreatment group. On the other hand, PSP94 expression was decreased in grade 3 tumor foci but increased in grades 4 and 5 tumor foci compared with samples of the corresponding grade in the no pretreatment group (p = 0.0034). CONCLUSIONS: PSP94 expression in benign prostate persists under androgen deprivation compared to PSA. PSP94 synthesis in high grade tumor appears to be activated in the absence of androgen stimulation, indicating the possible alternative pathways in the regulation of PSP94.

PSP94 (or beta-microseminoprotein) is a secretory protein specifically expressed and synthesized in the lateral lobe of the rat prostate .

BACKGROUND: Prostatic secretory protein of 94 amino acids (PSP94), also called beta-microseminoprotein, is a small, nonglycosylated protein, rich in cysteine residues. It was first isolated as a major protein from human seminal plasma. Subsequently, its homologous proteins were identified, and their cDNAs or genes have been cloned in primates, pigs, and rodents. METHODS: The present study investigated the expression pattern of PSP94 in the normal Noble rat prostate gland by nonradioactive in situ hybridization, Northern blotting, RT-PCR, Western blotting, and immunohistochemistry. Its expression in the mouse prostate gland was also examined by in situ hybridization. RESULTS: The results of in situ hybridization, and Northern and Western blot analyses, showed that the expression of rat PSP94 was prostate-specific. It was highly expressed in the lateral prostatic lobe, moderate in the dorsal lobe, weak in the coagulating gland, and negative in the ventral lobe and seminal vesicle. Its specific expression in the rat prostate gland was further confirmed by RT-PCR analysis of prostatic and nonprostatic organ tissues. Its mRNA transcripts were not detected in the urinary, digestive, and respiratory tracts, male and female reproductive organs, muscles, brain, and kidney. Its molecular mass was estimated to be 14.5 kDa by Western blotting. Similar prostate-specific expression of PSP94 was also observed by in situ hybridization in the lateral lobe, but not in the dorsal and ventral lobe, of the mouse prostate gland. CONCLUSIONS: Rat PSP94 is a major secretory protein highly expressed and synthesized by the lateral lobe of both rat and mouse prostate glands, and moderately expressed in the dorsal lobe of the rat prostate gland.

Serum bound forms of PSP94 (prostate secretory protein of 94 amino acids) in prostate cancer patients.

PSP94 (prostate secretory protein of 94 amino acids) was regarded as a possible prostate cancer marker, however, it has been controversial. All prior studies were designed to test the free form in serum using antibodies to PSP94. Results presented here demonstrate that PSP94 exists in prostate cancer patients in two forms, free and bound, and that the majority is present as serum bound complexes. This result was demonstrated by using both native and SDS-PAGE analyses of serum proteins from prostate cancer patients. Chromatographic separation of serum total proteins by a molecular sieve column generated two peaks (peak I and II), which were reactive with rabbit antiserum to human PSP94 in Western blot experiments. Peak I was eluted before the IgG fraction at a molecular weight larger than 150 kDa, and peak II appeared after serum albumin ( approximately 67 kDa) was eluted. By using a biotinylated PSP94 as an indicator of the free form of PSP94, we demonstrate that peak I contains serum PSP94-bound complexes and peak II is likely the free form of serum PSP94. Since the molecular weight of serum PSP94-bound complexes is close to IgG during molecular sieve separation, serum PSP94 complexes were further purified through two rounds of protein A column separation, followed by DEAE-ion exchange column chromatography. In vitro dissociation tests of the purified PSP94-bound complexes showed that the binding of serum PSP94-complexes is probably via disulfide bonds and is chemically stable. The results presented here indicate that serum PSP94-bound complexes must be considered in evaluating the clinical utility of PSP94 as a prostate cancer marker.

In situ hybridization study of PSP94 (prostatic secretory protein of 94 amino acids) expression in human prostates.

BACKGROUND: The prostatic secretory protein of 94 amino acids (PSP94), also named beta-microseminoprotein, is one of the major proteins secreted by the human prostate. However, its value as a prognostic marker for prostate cancers is still under debate. The aim of the present study was to examine the expression pattern of this protein in fetal, pubertal, and aged human prostates. METHODS: Nonisotopic in situ hybridization using a digoxigenin-labeled riboprobe for PSP94 and immunohistochemistry were used to demonstrate the expression of PSP94 in different regions or zones of fetal, pubertal, and adult human prostates. Its localization pattern was also compared with those of two other major secretory proteins, prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP), by immunohistochemistry. RESULTS: PSP94 mRNA and its protein were localized to the secretory epithelium of normal pubertal and adult human prostates. No hybridization signal and immunoreactivity of PSP94 were seen in fetal prostates at 6-7 months of gestation, whereas some glandular cells were positive to PSA and PAP immunostainings. In the adult prostates, PSP94 expression was intense in the acini in the peripheral zone, less intense in the transition zone, and variable in the central zone. Such a zonal expression pattern was more apparent in the pubertal prostates. However, no obvious differential expression pattern was observed in the immunohistochemistry of PAP and PSA, which showed a uniform staining of the secretory epithelia of the acini in all anatomic zones. The hybridization signals and immunoreactivity of PSP94 became reduced or lost in premalignant prostatic intraepithelial neoplastic lesions and different grades of prostatic carcinomas. CONCLUSIONS: Fetal prostates at 6-7 months of gestation already synthesize PSA and PAP but not PSP94. The delayed expression of PSP94 appears to correlate with the development of the prostate gland. A differential expression pattern of PSP94 is demonstrated in different anatomical zones, showing that this protein is more expressed and synthesized in the acini in the peripheral zone than in the central and transition zones. However, such a zonal pattern is not seen in the immunohistochemistry of PSA and PAP. The present study also shows that PSP94 is downregulated in different grades of prostate cancers.

Differential expression of PSP94 in rat prostate lobes as demonstrated by an antibody against recombinant GST-PSP94.

Prostate secretory protein (PSP94, 94 amino acids) is one of the most abundant proteins secreted from the prostate. Its biological role is unknown and still controversial, although it is assumed to have the potential to be a biomarker and a suppressor of prostate cancer. In order to establish an animal model to further elucidate its biological role, we expressed the mature form of rat PSP94 in Escherichia coli, using a glutathione S-transferase (GST) fusion expression vector; we generated a polyclonal rabbit antibody against the recombinant protein. The antibody specifically recognized recombinant rat PSP94 and cross-reacted only very weakly with its human homologue. Using the characterized anti-rat PSP94 antibody, we found that PSP94 was located primarily in rat prostate. Furthermore, PSP94 is present at different levels in different lobes of rat prostate, with significant levels detectable only in the lateral lobe (LP). In addition, the most abundant PSP94 expression was found in the prostate lobe secretions, and PSP94 levels in LP secretions were at least seven times higher than in secretions from the dorsal prostate (DP). The rat ventral prostate (VP) and other regions of the male accessory glands were found to be almost completely devoid of PSP94. Since most rat prostate dysplasia induced by steroid hormone treatment occurs only in dorsolateral prostate, prostate tissue-specific expression and the expression of PSP94 in dorsolateral, but not other, lobes of the prostate suggest a potential role in prostate targeting and prostate cancer development.

Prognostic significance of beta-microseminoprotein mRNA expression in prostate cancer.

BACKGROUND: Human beta-microseminoprotein (beta-MSP or PSP94) is a small protein secreted by prostatic epithelial cells. We recently reported the presence of low levels of beta-MSP mRNA expression and protein in most prostate cancer tissues. METHODS: Beta-MSP and mRNA expression was examined by in situ hybridization in biopsy specimens obtained from 92 patients with prostate cancer. All tissue specimens were obtained by needle biopsies prior to treatment. All patients subsequently received endocrine therapy. To estimate the influence of beta-MSP mRNA expression and three possible prognostic factors, i.e., patient age, clinical stage, and Gleason score, on time to progression under endocrine therapy, univariate and multivariate analyses were performed using Cox's proportional hazards regression model. RESULTS: Multivariate survival analysis showed that clinical stage was the strongest prognostic factor (P = 0.006) and that beta-MSP mRNA expression was the second strongest factor (P = 0.038) in 92 patients with stage B-D disease. Analysis of only 51 patients with stage D disease showed that beta-MSP mRNA expression was the only significant prognostic indicator for progression under endocrine therapy (P = 0.003). CONCLUSIONS: The presence of cells that express the beta-MSP transcript may be a novel indicator of potentially aggressive prostate cancer.

cDNA, genomic cloning, and gene expression analysis of mouse PSP94 (prostate secretory protein of 94 amino acids).

The potential use of prostate secretory protein of 94 amino acids (PSP94) as a diagnostic biomarker or a therapeutic agent for prostate cancer has been reported. In order to establish an animal model to further elucidate on its biological role, we cloned the mouse PSP94 cDNA (approximately 500 bp) by reverse transcriptase-polymerase chain reaction (RT-PCR) and disclosed its genomic structure. The whole mouse PSP94 gene (approximately 23 kb) was amplified by long and accurate-PCR and also cloned by screening of a mouse embryo stem-cell genomic library. Computational and statistical analyses have demonstrated several highly conserved characteristics of PSP94 among different species. Comparison of PSP94 from human, two primates, pig, and rodents revealed that the most significant feature is that PSP94 is rich in cysteines (10% of the total sequence) and their positions are highly conserved. The three intron-four exon structure of the human PSP94 gene and the consensus sequence (....GT-intron-AG...) for mRNA splicing are also strongly conserved. A high divergence in cDNA sequence in the protein-coding region and also in the genomic sequence of PSP94 was also observed among these species. Comparing with alpha-globin, a typical evolutionally conserved gene, with the PSP94 gene, the rate of nonsynonymous changes per site per year (kN) is 2 to 6 times higher, indicating that PSP94 gene has been under far fewer evolutionary constraints than other genes and has a potential role as a species barrier in reproductive biology. In order to test this hypothesis, we investigated the gene expression of PSP94 and its tissue distribution in various rodent tissues by RT-PCR and in situ hybridization (ISH). Gene expression was found only in the prostate, suggesting that PSP94 is probably more tissue specific in the prostate of rodents than in mammals. The ISH analysis also revealed a prostate lobe-specific expression of the PSP94 gene in both mice and rats. It was strongly expressed in the lateral prostate, but the findings were negative in the dorsal and ventral lobe. Therefore, it is hypothesized that one of the primary functions of rodent PSP94, as a major prostate secretory protein, is related to reproductive biology.

Osteopontin inhibits inducible nitric oxide synthase activity in rat vascular tissue.

We tested the hypothesis that osteopontin (OPN) can inhibit the induction of inducible nitric oxide synthase (iNOS) in vascular tissue. iNOS activity was induced in rat thoracic aortas by incubation of the tissue with lipopolysaccharide (LPS) and measured by conversion of L-[3H]arginine to L-[3H]citrulline. Addition of >/=1 nM recombinant OPN protein significantly reduced the LPS-induced increase in iNOS activity. Western blotting and the RT-PCR were used to determine the effect of LPS with and without OPN on tissue levels of iNOS protein and RNA, respectively. LPS resulted in an increase in iNOS protein and RNA, whereas OPN dose-dependently reduced tissue levels of iNOS activity, protein, and RNA. Mutated OPN proteins, in which the integrin-binding RGD amino acid sequence was deleted or mutated to RGE, resulted in complete and partial loss, respectively, of the ability of OPN to inhibit LPS-induced iNOS activity, implicating integrin binding in the effect. These results indicate that OPN can prevent induction of iNOS in vascular tissue.

MMP-2 expression is associated with, but not sufficient for, malignant conversion of murine LTA cells.

We previously showed that tumorigenic, non-metastatic LTA cells can be converted to a metastatic phenotype either by cell fusion with non-malignant NIH 3T3 cells, or by transfection with genomic DNA from metastatic murine B16F1 or human 1GR37 melanoma cells. In order to identify a gene present in NIH 3T3 cells that is responsible for this conversion, we transferred DNA from an NIH 3T3 genomic library into LTA cells and tested for changes in metastatic properties, assessed in the chick embryo. We found that 3 of 4 pools of transfectant clones showed significantly increased metastatic ability over the vector-only control transfectants. All three metastatic transfectant pools showed significantly increased RNA levels of the 72 kDa type IV gelatinase (MMP-2). To test whether increased expression of MMP-2 was sufficient to convert LTA cells to metastatic ability, we transfected full length MMP-2 cDNA, in a CMV-promoter expression construct, into LTA cells. Stable transfectants with elevated MMP-2 RNA and enzymatic activity were obtained. The highest MMP-2 expressing clone was assayed for experimental metastatic ability in the chick embryo, and found to be no more metastatic than LTA parental cells. We conclude that increased MMP-2 expression accompanies the malignant conversion of LTA cells, but MMP-2 expression alone is not sufficient to bring about this change. The inability of LTA cells to metastasize thus appears to be due to a more complex defect than insufficient MMP-2. This study supports the idea that malignant conversion may require the concerted activation of multiple genes, which are in turn controlled by regulatory genes, whose identification will be important in understanding and controlling metastasis.

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (I). Immuno-dominant and immuno-recessive area.

PSP94 is a potential biomarker for evaluating patients with prostate carcinoma. We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94. Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N-terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C-terminus are completely devoid of antigenic activity with the polyclonal antibody. These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N-terminal peptides (N30 and M23) v. either recombinant GST-PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein. To further delineate the antigenic activity of the N- and C-termini, we have also expressed N- and C-terminal half of the whole PSP94 (each 47 peptides) using the E. coli GST expression system. The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments. Dose response of the recombinant GST-PSP-N47 and -C47 peptides to PSP94 polyclonal antibody showed differential binding activities. Competitive binding of these recombinant N47/C47 proteins against the GST-PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide. Based on the immunological studies of both synthetic peptides and recombinant PSP94- N/C terminal proteins, we propose an epitope structure of human PSP94 with an immuno-dominant N-terminus and an immuno-recessive C-terminus.

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (II). Epitope mapping by monoclonal antibodies.

PSP94 has shown potential to be a serum biomarker for evaluating prostate cancer. Studies of the epitope structure is crucial for this endeavour. In this article, we have used 15 different monoclonal antibodies (MAb) to analyse the epitope structure of PSP94 and to compare with the results obtained from our previous work using polyclonal antibody and recombinant PSP94. Firstly, we determined the relative activities of the 15 MAb population by direct and competitive ELISA. The two predominant MAbs (MAb PSP-6 and -19) in 15 MAbs were selected for further studies of the epitope structure. By comparing the binding activities of recombinant GST-PSP94 and natural PSP94 with MAbs, and by comparing their affinity with MAbs in an in vitro denaturing experiment, PSP94 was shown to have a similar, prevalently linear epitope structure as we demonstrated by polyclonal antibody. Using recombinant GST fusion protein with PSP94 and with each half of the N- and C-terminal 47 amino acids (GST-PSP-N47/C47) in E. coli cells, the different epitopes recognized by 15 monoclonal antibodies were delineated and the polar distribution of the epitope structure of PSP94 was characterized. Results of direct ELISA of recombinant N47 and C47 and their competitive binding against natural PSP94 (competitive ELISA) showed that the N- and C-termini represent the immuno-dominant and immuno-recessive area separately. A majority of the monoclonal antibodies (12/15) showed preferential binding of the N-terminal sequence of the PSP94 protein. Using GST-PSP-N47 as a standard protein, an epitope map of the 15 monoclonal antibodies was obtained. The results of this study will help to define the clinical utility of PSP94.