Åke Pousette

[4] Estimation of biomass in growing cell lines by adenosine triphosphate assay

Publisher Summary This chapter describes the steps involved in estimation of biomass in growing cell lines by luminometric ATP assay. Methods for studying the reliability of the results are described. In particular, estimation of energy charge (EC) by a new method for assay of ATP, ADP, and AMP was found to be valuable. The activity of several adenosine nucleotide converting enzymes as a function of the energy charge—that is, (ATP + 0.5 ADP) / (ATP + ADP + AMP)—indicates that the energy charge in intact metabolizing cells should be stabilized in the interval 0.8–0.9. By measuring all three adenine nucleotides, the measure of the physiological status of the cells is obtained that provides information on whether ATP can be used to estimate biomass or if the energy metabolism is disturbed. By the method described in the chapter, all three adenine nucleotides are determined in a single aliquot from the cell extract. Measurement of energy charge gives a reliable estimation of the accuracy of using ATP as a biomass parameter. Growth monitoring by assay of adenine nucleotides includes the following steps: (1) cultivation of cells, (2) sampling and pretreatment of samples, (3) extraction of adenine nucleotides from cells, (4) luminometric assay of adenine nucleotides, and (5) correlation to other biomass estimations.

Parenteral Estrogen versus Combined Androgen Deprivation in the Treatment of Metastatic Prostatic Cancer - Scandinavian Prostatic Cancer Group (SPCG) Study No. 5

Objective : In the mid-1980s, interest in parenteral estrogen therapy for prostate cancer was renewed when it was found that it influenced liver metabolism only marginally and had very few cardiovascular side-effects. In this study high-dose polyestradiol phosphate (PEP; Estradurin ® ) was compared to combined androgen deprivation (CAD) for the treatment of patients with metastatic prostate cancer. The aim of the study was to compare anticancer efficacy and adverse events, especially cardiovascular side-effects. Material and Methods : A total of 917 patients with T0-4, NX, M1, G1-3 prostate cancer and an Eastern Cooperative Oncology Group performance status of 0-2 were randomized to treatment with either PEP 240 mg i.m. twice a month for 2 months and thereafter once a month or flutamide (Eulexin ® ) 250 mg t.i.d. per os in combination with either triptorelin (Decapeptyl ® ) 3.75 mg per month i.m. or, on an optional basis, bilateral orchidectomy. A total of 556 patients had died at the time of this analysis. Results : There was no difference between the treatment arms in terms of time to biochemical or clinical progression and overall or disease-specific survival. There was no increase in cardiovascular mortality in the PEP arm. The PEP group had a higher prevalence of cardiovascular disease prior to the study and a significantly higher incidence of non-fatal ischemic heart events and heart decompensation during the study. Conclusions : PEP has an equal anticancer efficacy to CAD and does not increase cardiovascular mortality. Final evaluation of cardiovascular morbidity is awaiting further analysis and follow-up. PEP is considerably cheaper than CAD.

Androgen Induction of Prostate Cancer Cell Invasion Is Mediated by Ezrin

Ezrin is a key signaling molecule that regulates cell survival, adhesion migration, and invasion. We have previously shown that ezrin is regulated by androgen in rat prostate and that its expression is increased in prostate cancer and in prostate intraepithelial neoplasia. We have used the androgen-sensitive cell line LNCaP-FGC to investigate the role of ezrin in androgen-induced cell invasion. We found that androgen treatment of LNCaP-FGC cells induces ezrin expression, an effect that is inhibited by the androgen receptor antagonist, bicalutamide. In addition, androgen treatment induces the phosphorylation of ezrin in Thr-567 and Tyr-353 in a sequential manner. This is mediated through protein kinase C α and Src tyrosine kinase, respectively. Androgen treatment induces the translocation of both protein kinase C α and ezrin to the cell membrane and their association. Inhibition of ezrin function using short interference RNA or the overexpression of T567A and Y353F-ezrin mutants significantly reduces androgen-induced Matrigel invasion but does not affect cell proliferation or cell adhesion. Matrigel invasion of the androgen-insensitive prostate cancer cell lines PC-3 and LNCaP-R is also dependent on ezrin. In summary, we have shown that androgens regulate ezrin at transcriptional and posttranscriptional levels. Hormonal regulation of ezrin phosphorylation is required for androgen-induced cell invasion.

Inhibition of steroid sulfatase activity by danazol.

Abstract. The hydrolysis of dehydroepiandrosterone sulfate (DHAS) by human liver cells in culture and the hydrolysis of and formation of estradiol‐17β (E2) from estrone sulfate PIS) by human breast tumor preparations in vitro were studies in the presence and absence of danazol. In the latter tissue the effects of medroxyprogesterone acetate (MPA), trilostane, aminoglutethimide (AG) and tamoxifen were tested for comparison. Danazol at concentrations of 1.4 × M strongly inhibited the hydrolysis of DHAS as well as the hydrolysis of and formation of E2 from E1S. With the exception of a slight inhibitory effect of trilostane upon E1S hydrolysis, the other four drugs did not inhibit the metabolism of E1S. Danazol, at concentrations corresponding to those occurring in vivo during therapy, is a potent inhibitor of steroid sulfatase activity. This may be one of the ways in which the drug affects peripheral and target tissue levels of steroid hormones.

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-3β 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.

Cortisol and androgen concentrations in female and male elite endurance athletes in relation to physical activity

SummaryTo evaluate the effect of variations in physical activity on selected hormone concentrations in male versus female athletes, fasting serum concentrations of cortisol (C), total-testosterone, free-testosterone, non sex hormone binding globulin (SHBG) bound testosterone, dehydroepiandrosterone, 4-androstene-3,17-dione and SHBG were studied. The tests were performed in nine male and seven female elite endurance athletes during the off-season (test 1), early in the competition season (test 2) and at the end of the competition season (test 3). The C concentration increased significantly during the competition season in women but not in men. Further, the mean C concentrations at test 3 as well as the mean level during the whole observation period (tests 1, 2, 3) were significantly higher in women than in men. No significant changes were found in androgen concentrations or androgen: cortisol ratios within the two groups. The differences between the sexes in C response may indicate different adaptive mechanisms to similar physical stress.

Proteomic comparison of prostate cancer cell lines LNCaP-FGC and LNCaP-r reveals heatshock protein 60 as a marker for prostate malignancy.

Androgen‐sensitive prostate cancer cell‐line LNCaP‐FGC and androgen‐resistant line LNCaP‐r constitute a model for development of androgen resistance in prostate cancer.

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

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.

Deoxyribonucleic Acid Ploidy and the Direct Assay of Prostatic Acid Phosphatase and Prostate Specific Antigen in Fine Needle Aspiration Biopsies as Diagnostic Methods in Prostatic Carcinoma

We used fine needle biopsies from prostatic tumors at routine examinations in 133 patients. Cytological grading was performed with a scoring system. Cellular prostatic acid phosphatase and cellular prostate specific antigen from the aspirates were quantitated. Deoxyribonucleic acid flow cytometry was performed and the tumors were subdivided into diploid, tetraploid and aneuploid groups. Tumor staging was assessed by digital examination. A decrease in the biochemical markers was significantly correlated with the increase in malignancy grade, tumor stage and a shift from diploid to aneuploid tumors. Cellular prostatic acid phosphatase and cellular prostate specific antigen as well as tumor ploidy may contribute to the objective determination of the malignancy potential of the prostatic carcinoma.

Quantitative Estimation of Tissue Prostate Specific Antigen, Deoxyribonucleic Acid Ploidy and Cytological Grade in Fine Needle Aspiration Biopsies for Prognosis of Hormonally Treated Prostatic Carcinoma

The prognostic value of deoxyribonucleic acid (DNA) flow cytometry, cytological grading and the direct assay of prostate specific antigen (PSA) in the material of fine needle aspirates was studied in 67 consecutive patients with newly detected prostatic carcinoma. All patients were hormonally treated (castration in 27 and luteinizing hormone-releasing hormone agonist or parenteral estrogens in 40). The patients were followed for a minimum of 2 years. PSA was analyzed in the biopsy material by a direct radioimmunoassay and related to the total amount of DNA. In parallel biopsies DNA ploidy using flow cytometry and cytological grade were established. Patients with a geometric mean value of greater than or equal to 0.12 microgram. PSA/microgram. DNA had a progression rate of 7%, compared to 59% for those with less than 0.12 microgram. PSA/microgram. DNA. In Cox multivariate analysis cytology and tissue PSA content were the most important factors in expressing the difference for interval to progression in hormonally treated patients.