Ezrin mediates c-Myc actions in prostate cancer cell invasion.

The forced overexpression of c-Myc in mouse prostate and in normal human prostate epithelial cells results in tumor transformation with an invasive phenotype. How c-Myc regulates cell invasion is poorly understood. In this study, we have investigated the interplay of c-Myc and androgens in the regulation of prostate cancer cell invasion. We found that c-Myc induces cell invasion and anchorage-independent growth by regulating ezrin protein expression in the presence of androgens. The activity of the ezrin promoter is controlled by androgens through c-Myc, which binds to a phylogenetically conserved E-Box located in the proximal promoter region. Besides, we also show that ezrin is an important regulator of c-Myc protein levels. These effects are achieved through androgen-induced changes in ezrin phosphorylation, which results in the regulation of downstream signals. These downstream signals involve the modulation of Akt and GSK-3beta activity resulting in increased c-Myc protein synthesis and inhibition of its degradation. In summary, we have shown a key role for ezrin as a mediator of c-Myc-induced tumorigenesis in prostate cancer cells.

Pharmacogenetics during standardised initiation of thiopurine treatment in inflammatory bowel disease.

BACKGROUND: Firm recommendations about the way thiopurine drugs are introduced and the use of thiopurine methyltransferase (TPMT) and metabolite measurements during treatment in inflammatory bowel disease (IBD) are lacking. AIM: To evaluate pharmacokinetics and tolerance after initiation of thiopurine treatment with a fixed dosing schedule in patients with IBD. PATIENTS: 60 consecutive patients with Crohn's disease (n = 33) or ulcerative colitis (n = 27) were included in a 20 week open, prospective study. METHODS: Thiopurine treatment was introduced using a predefined dose escalation schedule, reaching a daily target dose at week 3 of 2.5 mg azathioprine or 1.25 mg 6-mercaptopurine per kg body weight. TPMT and ITPA genotypes, TPMT activity, TPMT gene expression, and thiopurine metabolites were determined. Clinical outcome and occurrence of adverse events were monitored. RESULTS: 27 patients completed the study per protocol, while 33 were withdrawn (early protocol violation (n = 5), TPMT deficiency (n = 1), thiopurine related adverse events (n = 27)); 67% of patients with adverse events tolerated long term treatment on a lower dose (median 1.32 mg azathioprine/kg body weight). TPMT activity did not change during the 20 week course of the study but a significant decrease in TPMT gene expression was found (TPMT/huCYC ratio; p = 0.02). Patients with meTIMP concentrations >11,450 pmol/8 x 10(8) red blood cells during steady state at week 5 had an increased risk of developing myelotoxicity (odds ratio = 45.0; p = 0.015). CONCLUSIONS: After initiation of thiopurine treatment using a fixed dosing schedule, no general induction of TPMT enzyme activity occurred, though TPMT gene expression decreased. The development of different types of toxicity was unpredictable, but we found that measurement of meTIMP early in the steady state phase helped to identify patients at risk of developing myelotoxicity.

Androgens induce CD-9 in human prostate tissue.

Based on microarray analyses of LNCaP and LNCaP-r prostatic cell-lines we tentatively identified CD-9 as an androgen sensitive protein. This prompted us to characterize the androgen sensitivity and the correlation to malignancy of CD-9 at the protein level. Using Western blot, RT-PCR and immunohistochemistry the expression of CD-9 was analysed in LNCaP cells stimulated during increasing time by the synthetic androgen R1881 and also in 88 specimens of human prostate cancer tissues. Expression of CD-9 was induced by R1881 in LNCaP. CD-9 was immunolocalized in human prostate tissue sections representing non-malignant tissue as well as tumour areas. In non-malignant glands CD-9 immunoreactivity was observed at the apical and lateral cell borders of luminal epithelial cells. Basal epithelial cells were largely unstained. In tumour areas CD-9 staining intensity was variable and apparently not related to primary Gleason grade. In prostate tissue from a patient under androgen ablation therapy no staining was observed in luminal epithelial cells or in the tumour areas, but some staining was observed in basal epithelial cells. CD-9 is regulated by androgens in LNCaP and present in human prostate specimens. The expression is variable and changes in expression levels. These and earlier studies using other tissues indicate that CD-9 and its cellular localization could have an important role in prostate cancer cell development.

Tissue concentrations of tissue polypeptide antigen (TPA) and prostatic specific antigen (PSA) in 42 patients with prostatic carcinoma.

BACKGROUND: Following development of methods to quantitate biochemical markers in aspiration biopsies we showed that tissue concentration of prostate specific antigen (T-PSA) decreased with increasing malignancy while serum PSA increased. We also found that T-PSA predicts the clinical outcome better than earlier used prognostic markers. METHODS: In order to further study biochemical markers in prostatic cancer a membrane protein, tissue polypeptide antigen (TPA), which is a complex of polypeptide fragments of cytokeratins 8, 18, and 19, was quantitated in 42 patients with newly diagnosed carcinoma of the prostate. The samples had previously been analyzed for T-PSA. RESULTS: Correlation to TGM classification showed that higher malignancy is correlated to lower tissue TPA values. There is a significant positive correlation (r(s) = 0.49, P < 0.01) between T-TPA and T-PSA. Pretreatment values of T-PSA, but not T-TPA, had association to time to progression or time to death. CONCLUSIONS: Increasing prostatic malignancy is correlated to decreasing values of T-TPA. This indicates that the concentrations of membrane and secretory proteins are changed in the same direction in tissue during cancer development. Tissue TPA seem to have no prognostic value in endocrine treatment of prostatic carcinoma.

Estrogens increase the endothelial nitric oxide synthase (ecNOS) mRNA level in LNCaP human prostate carcinoma cells.

BACKGROUND: The endothelial cell-specific form of nitric oxide synthases (ecNOS) may play an important role in vascular development, maintenance of vascular tone, and tumor growth in human prostate cancer. Estrogens have been shown to upregulate ecNOS expression in different human cell culture systems. Estrone sulfate (E1S) is the most abundant circulating estrogen, and may serve as a prehormone for the terminal biologically active estrogen estradiol-17beta (E2) in men. METHODS: The effects of E1S and E2 on mRNA expression of ecNOS were studied in the androgen-sensitive LNCaP-FGC cell line and its androgen-resistant derivative, LNCaP-r. The cells were grown in steroid-depleted medium and incubated for 2-4 or 48 hr with 0-100 nM of E1S and E2, respectively. ecNOS mRNA levels were determined using RT-PCR and are expressed as arbitrary units after correction for control HGPRT gene mRNA levels. RESULTS: Treatment for 48 hr with 10 and 100 nM E1S significantly (P<0.05) increased ecNOS mRNA levels in LNCaP-FGC cells. Significantly higher (P<0.05) ecNOS mRNA levels also were found in LNCaP-FGC cells treated with E2 for 2-4 hr, irrespective of E2 concentration. The level of ecNOS mRNA was significantly lower (P<0.05) in untreated LNCaP-r than in LNCaP-FGC. LNCaP-r cells incubated with 100 nM E2 for 48 hr had a significantly higher (P<0.05) level of ecNOS mRNA than control LNCaP-r cells. CONCLUSIONS: The results indicate that ecNOS mRNA expression in LNCaP-FGC can be induced by E2, but also by its prehormone E1S, probably after conversion to E2. However, the different stimulation patterns observed for E2 and E1S in LNCaP-FGC and LNCaP-r cells also could indicate stimulatory as well as inhibitory effects of estrogens in this model system, and this could depend on time of exposure and the concentration of active estrogen.

Prognostic value of serial tissue prostate-specific antigen measurements during different hormonal treatments in prostate cancer patients.

To reveal the effects of different hormonal treatments directly on the prostate during treatment, the concentration of prostate-specific antigen in the tissue (T-PSA) was studied in 63 patients with untreated newly diagnosed carcinoma of the prostate (CaP). T-PSA measurements were performed in fine-needle aspiration biopsies at the time of diagnosis and 6, 12, and 24 months after initiation of treatment. Treatments modalities were bilateral orchidectomy, gonadotropin-releasing hormone (GnRH) agonists, or parenteral estrogens. Thirty-one (49%) of the patients died of CaP and 18 (29%) of other diseases. Fourteen of the patients (22%) were still alive at the end of the observation period (median follow-up time, 111.5 months; range, 98-128 months). In all of the 31 patients who died of CaP, T-PSA values increased during treatment. This increase was observed long before clinical signs of progression appeared (median of interval, 14 months). Twenty of these 31 patients showed an increase in T-PSA from pretreatment values at 6 months. At 12 months this increase was observed in 30 of 31 patients. In contrast, in all of the patients who responded to the hormonal regimen, T-PSA values decreased and remained low during treatment. Furthermore, the patients who did not die of CaP and received estrogen treatment had significantly higher T-PSA values compared with those who were treated with bilateral orchidectomy or GnRH agonists. This indicates that estrogens may stimulate PSA synthesis in tumor tissue in vivo in the presence of castration levels of testosterone. Statistical evaluation showed that the T-PSA ratio between month 12 and month 0 had the most significant prognostic value for predicting the clinical outcome. This ratio was superior to clinical classifications, e.g., tumor stage and cytological grade, and also was higher than T-PSA at the time of diagnosis. This study has shown that aspiration biopsy material can be used to reveal biochemical changes in the tissue during treatment and that one specific marker (T-PSA) can predict the clinical outcome of endocrine treatment of CaP patients better than previously used methods. We believe that selected tissue markers or the protein pattern can help us to characterize the tumors and predict the clinical outcome so an optimal treatment can be chosen for every patient.

Prognostic significance of tissue prostate-specific antigen in endocrine-treated prostate carcinomas.

Fine-needle aspiration biopsy is a minimally invasive technique for obtaining sample material suitable not only for cytological grading but also for flow cytometry and for biochemical analyses. The prognostic value of tissue prostate-specific antigen (T-PSA) from fine-needle aspiration biopsies was compared with serum total and free prostate-specific antigen, the ratio of free:total serum prostate-specific antigen, tumor stage, cytological grade, and DNA ploidy in 179 patients with stage T2-T4 prostate cancer (CAP). The patients, who were free from bone metastases at the time of diagnosis, were treated by either orchidectomy or medical castration with GnRH analogues or high-dose parenteral depot estrogens. They were followed for at least for 71 months or until death, and the different variables were correlated to time to progression and time to death from CAP. Using Cox univariate analysis, T-PSA was shown to be the most important factor in predicting time to progression and time to death. When the patients were divided into three groups with respect to T-PSA, 56 of 60 (93%) of the patients with low T-PSA levels developed progressive disease, and 52 of 60 (87%) died of CAP. For patients with intermediate T-PSA levels, the corresponding figures were 9 of 60 (15%) and 6 of 60 (10%). None of the 59 patients with high T-PSA values developed progressive disease. Similar but less pronounced relationships were found between tumor progress and CAP-specific death on the one hand and clinical stage, cytological grade, and DNA ploidy on the other. In a Cox multivariate stepwise analysis, T-PSA was the only important factor for time to progression and death. This was also true for the subgroup of patients with stages T2 and T3 disease only. The study shows that T-PSA is superior to other hitherto routinely used markers for the prediction of outcome of hormone-treated patients with newly diagnosed CAP.

Time for revival of estrogens in the treatment of advanced prostatic carcinoma? Pharmacokinetics, and endocrine and clinical effects, of a parenteral estrogen regimen.

BACKGROUND: The present pilot study tested the clinical performance of a new pharmacokinetically guided dosing regimen of parenteral estrogen in patients with advanced prostatic carcinoma. The aim was to accelerate endocrine effects and to avoid cardiovascular side effects. METHODS: Seventeen patients were randomized to intramuscular injections of 240 mg polyestradiol phosphate (PEP) every second week for the first 8 weeks (five doses), followed by a maintenance dose of 240 mg every month; and 16 patients were randomized to bilateral orchidectomy. The estrogen dosing was calculated by pharmacokinetic modelling to achieve a rapid increase in serum estradiol and thereby a fast decrease in testosterone. RESULTS: The predicted increment in serum estrogen was achieved, together with a subsequent decrease in testosterone in the PEP group. In addition, there were no signs of an increased cardiovascular morbidity. This was probably due to a minimal estrogenic influence on the liver and was reflected by unchanged levels of coagulation factor VII. Clinical effects, during the first 2 years of treatment, were similar in the two treatment arms, with 12 patients in the orchidectomy group and 14 patients in the PEP group responding to treatment. CONCLUSIONS: The present parenteral regimen is an efficient and time-saving estrogen regimen with a favorable side-effect profile. PEP seems to offer a potential for revival of the most cost-effective endocrine treatment of cancer of the prostate, i.e., estrogen.

Tissue PSA is the best predicting variable for the outcome of endocrine treatment of prostatic carcinoma.

In order to evaluate the prognostic value of tissue-PSA (prostatic-specific protein, measured in aspiration biopsies) 231 hormonally treated patients with verified carcinoma of the prostate (CaP) but without metastasis were studied retrospectively. T-PSA was determined at the time of diagnosis in all patients and in 52 of these also at 6, 12 and 24 months after diagnosis. Of the 231 patients, 79 died of prostatic carcinoma and 152 were still alive or had died of other diseases at the end of the observation period (more than 71 months). In a first set of evaluations the predictive value of a single analysis at the time of diagnosis was studied in 179 patients. It was found that tissue PSA was the most important factor for predicting both time to progression and time to death in CaP. Other competing factors were S-PSA, free S-PSA, age, clinical stage, grade and DNA-ploidy. Further evaluations regarding serial PSA determinations were performed in 52 patients. Tissue PSA increased during treatment in all patients who died of CaP. In all patients who survived or died for other reasons, tissue PSA decreased during treatment and remained low. The change of tissue PSA seen between 0, 6 and 12 months could in all cases predict the clinical outcome. It is concluded that a single analysis of tissue PSA at the time of diagnosis can predict the clinical outcome in most cases and that serial determinations can predict the outcome in almost all cases of a CaP without metastasis at the time of diagnosis. This requires that we use this assay when selecting between patients who will survive on hormonal treatment and those who most probably would benefit from a more aggressive treatment.

Tissue PSA from fine-needle biopsies of prostatic carcinoma as related to serum PSA, clinical stage, cytological grade, and DNA ploidy.

BACKGROUND: The mechanisms behind changes in serum PSA (S-PSA) levels in patients with prostatic carcinoma (CAP) are not completely known. To further elucidate the factors affecting the serum levels of this important tumor marker, we measured PSA concentrations in serum and in aspiration biopsies (tissue PSA; T-PSA) from patients with prostatic disease and correlated the values to tumor stage, cytological grade, and DNA ploidy. METHODS: T-PSA and S-PSA were measured in 91 metastasis-free patients with newly diagnosed, untreated CAP and 13 patients with benign prostatic hyperplasia, and the values were related to tumor stage, cytological grade, and DNA ploidy. RESULTS: Significant negative correlations were found between T-PSA and S-PSA in the total clinical material and various subgroups of patients with CAP. T-PSA showed significant negative associations to T-stage and to cytological grading, and T-PSA concentrations were significantly lower in tetra-/aneuploid tumors than in diploid tumors. On the other hand, S-PSA showed corresponding positive associations and was significantly higher in tetra-/aneuploid tumors than in diploid tumors. CONCLUSIONS: The negative association between S-PSA and T-PSA values indicates that S-PSA values in metastasis-free patients reflect the degree of leakage from the tumor tissue rather than the intracellular concentration of PSA. Factors such as tissue volume, condition of gland structure, and vascularization may thus be more important for S-PSA than the production of PSA in the prostatic tissue.

Free and total prostate-specific antigen serum concentrations do not help to detect prostate cancer in patients with urinary outlet obstruction.

OBJECTIVES: To determine whether different molecular forms of prostate-specific antigen (PSA) obtained before transurethral resection of the prostate (TURP) indicate the presence of prostate cancer. PATIENTS AND METHODS: The free, total and free-to-total PSA levels were measured in 261 patients scheduled for TURP, 20 of whom had known prostate cancer. The tissue histology was compared with the PSA levels and the patients were followed for 5 years. RESULTS: Prostate cancer was detected in 23 of the patients (9%) who were thought to have benign disease. Normal ranges for the distribution of the PSA levels were established based on the patients with a benign histology, but these ranges did not detect most of the unknown cancers. The sensitivity of the total PSA test in detecting cancer was 38% and the specificity 90%. The discrimination was no better when considering the free fraction or the free-to-total PSA level. However, none of the 14 patients whose cancer was missed showed general progression of the disease during the 5-year follow-up and only one died from prostate cancer. In contrast, eight of the 20 patients with a known prostatic malignancy showed general progression, and six died from the disease. CONCLUSION: PSA testing of patients with outlet obstruction often failed to detect prostate cancer, but the prognosis was moderately good in those patients in whom it was missed.

Serum FSH levels in women with polycystic ovary syndrome during ovulation induction using down-regulation and urofollitropin.

OBJECTIVE: To evaluate retrospectively the use of serum FSH levels and to correlate them with follicular growth in a clinical ovulation induction program. METHODS: Twenty women with infertility due to anovulation associated with polycystic ovary syndrome (PCOS) were studied. The patients were down-regulated with a long GnRH agonist protocol and stimulated with purified urofollitropin, using a low-dose step-up regimen. Repeated serum samples were drawn and transvaginal ultrasound scans were-performed. During the exogenous FSH therapy serum FSH levels resulting in continuous follicular growth were analyzed, as well as the rates of ovulation, pregnancy, cancellation and conversion to in vitro fertilization (JVF). RESULTS: Thirty-two out of fifty treatment cycles led to ovulation, resulting in five term pregnancies. Eight cycles were converted to IVF/embryo transfer due to multiple follicular growth. They resulted in two pregnancies. Ten cycles were cancelled because of impaired follicular growth. The serum FSH levels (median 6 IU/I) resulting in continuous growth of the follicles were relatively stable within patients (variation 15%) but varied considerably between patients (45%). The relationship between FSH dose and serum level was different for lean and obese PCOS patients after subcutaneously injected urofollitropin CONCLUSIONS: There seems to be a difference in resorption/metabolism between lean and obese PCOS patients with regard to s.c. injected FSH. The intra-patient coefficient of variation (C.V.) of the serum FSH response level was quite low, as was the C.V. of the FSH dose at the response level. This allowed a more rapid dose adjustment in subsequent cycles. Analysis of serum FSH during induction of ovulation with gonadotropins seems to be of limited value in clinical programs.

Possible bone-preserving capacity of high-dose intramuscular depot estrogen as compared to orchidectomy in the treatment of patients with prostatic carcinoma.

BACKGROUND: Treatment of prostatic disease with GnRH agonists or by orchidectomy affects bone mass negatively. Estrogen treatment has beneficial effects on bone mass in women and might hypothetically have a bone preserving capacity also in patients with prostatic cancer. METHODS: We followed serum markers for bone and collagen metabolism and sex steroids for 18 months in patients with prostatic cancer treated by orchidectomy (N = 13) or by single-drug parenteral polyestradiol phosphate (240 mg intramuscularly every second week for the first two months, and then every fourth week; N = 17). RESULTS: Total and free testosterone reached castration levels within 1.5 months of estrogen treatment. Four patients developing progressive disease and/or signs of metastasis were excluded from the analysis. In the remaining patients, serum osteocalcin, procollagen IIIP (PIIINP), procollagen (PICP), and the crosslinked carboxyterminal telopeptide of type I collagen (ICTP) increased significantly over time following orchidectomy (N = 11). Serum osteocalcin and PICP decreased significantly over time during estrogen treatment (N = 15). Treatment values of all four markers were significantly lower in estrogen-treated than in orchidectomized patients. CONCLUSIONS: The changes in serum bone and collagen markers indicate an increased bone turnover in orchidectomized subjects. The opposite pattern was found in the estrogen-treated patients, indicating a reduced turnover. Estrogens may also have a bone mass-preserving capacity in elderly males with prostatic cancer.

Use of a hormone-sensitive (LNCaP) and a hormone-resistant (LNCaP-r) cell line in prostate cancer research.

BACKGROUND: In order to develop a hormone-resistant analog to the hormone sensitive cell-line LNCaP, different methods were tried. METHODS: After almost one year of continuous culture in RPMI-1640, containing a low concentration of androgens, the hormone sensitive cell-line LNCaP became hormone resistant and was named LNCaP-r. RESULTS: We used the LNCaP/LNCaP-r model system in order to study methods for the parenteral treatment of prostate carcinoma with estrogens, to study the mechanism of action of estramustine, and to identify markers useful as predictive tests for prostate carcinoma. CONCLUSIONS: A model system made up of the hormone sensitive prostate LNCaP cell-line and the hormone resistant LNCaP-r subline was developed and characterized. This system is, despite a number of limitations, easy to use as a first step to test different hypotheses that can be studied further in later clinical trials.

Temporal expression of platelet-derived growth factor (PDGF)-A and its receptor in human preimplantation embryos.

In order to improve assisted fertilization in humans it is important to elucidate the mechanisms of control of growth and development in the early pre-embryo. Increasing evidence shows that growth factors are of importance for such control mechanisms. As platelet-derived growth factor (PDGF) has been shown to enhance growth in a number of tissues, it may also be important in human pre-embryo development. PDGF acts as a dimer (AA, BB or AB) through its receptors: alpha alpha, beta beta and alpha beta. In order to study the role of PDGF and its receptors, we have used reverse transcription-polymerase chain reaction (RT-PCR) to examine the presence of transcripts in human pre-embryos that were surplus from the in-vitro fertilization treatment of infertile couples. Transcripts for PDGF A were present in the oocyte, 8-cell, morula and blastocyst stages but not in the 4-cell stage. Transcripts for PDGF B were not detected at any stage. PDGF receptor (PDGFR)-alpha transcripts were found in the 4-cell, 8-cell and blastocyst stages but not in the oocyte or morula stages. Transcripts for PDGFR-beta were detected from the 8-cell, morula and blastocyst stages but not in the oocyte or 4-cell stages. These results show that mRNA synthesis of both PDGF A and the two receptor subunits alpha and beta takes place from the 8-cell stage onwards, suggesting an autostimulatory pathway as a possible mechanism for growth factors during pre-embryo development.

Pharmacokinetics and testosterone suppression of a single dose of polyestradiol phosphate (Estradurin) in prostatic cancer patients.

The pharmacokinetics and endocrine effects of polyestradiol phosphate (PEP; Estradurin) were studied by determination of the concentrations of estradiol (E2), unconjugated (E1) and total estrone (tE1; > or = 85% estrone sulfate), and testosterone in serum from 11 prostatic cancer patients after administration of a single intramuscular injection (320 mg). After injection of PEP, serum concentrations of E2, E1, and tE1 increased during 2-3 weeks. Thereafter serum E2 declined monophasically with a mean half-life of 70 days. The elimination of E1 and tE1 seemed to be governed by the formation of E2. The testosterone concentration decreased inversely to the raising E2 level and reached castration levels within 3 weeks and remained at this level for about 2 weeks, whereafter it increased inversely to the decreasing E2 concentrations.

[Intramuscular depot estrogens (Estradurin) in treatment of patients with prostate carcinoma. Historical aspects, mechanism of action, results and current clinical status]. / Intramuskuläres Depotöstrogen (Estradurin) in der Behandlung von Patienten mit Prostatakarzinom. Historische Aspekte, Wirkungsmechanismus, Resultate und aktueller klinischer Stand.

More than 50 years ago, orally given estrogen was already used in the treatment of prostate cancer. Due to cardiovascular side-effects with a high morbidity of 25%, this treatment has not become standard. Recent investigations show that parenteral application reduces the risk of cardiovascular side-effects, because it avoids the first passage through the liver with high concentrations of estrogen which normally occur after oral application. Therefore, an increased synthesis of so-called "steroid-sensitive" liver proteins, such as coagulation factors (especially factor VII) can be avoided. This newer parenteral estrogen application shows encouraging results of a cheap and effective hormonal therapy with a low rate of side-effects in patients with prostate cancer.