Lynn N. Thomas

Androgen-Induced Regrowth in the Castrated Rat Ventral Prostate: Role of 5α-Reductase1

Testosterone (T), the major circulating androgen, must be converted to dihydrotestosterone (DHT) by the enzyme 5α-reductase (5α-R) to be maximally active in the prostate. The present study was designed to determine the relative potency of T and DHT on regrowth of the involuted prostate and to elucidate the role of 5α-R in the growing prostate. To create dose-response curves for intraprostatic T or DHT, rats were castrated for 2 weeks to allow their prostates to fully regress and then given T implants of various sizes in the presence or absence of the 5α-R inhibitor, finasteride. Markers for androgen effects on regrowth of the prostate were prostate weight, duct mass (a measure of secretory activity) and DNA content (a measure of cell number). To assess the relative uptake of T and DHT by the prostate, a comparison was made of intraprostatic DHT levels resulting from T and DHT implants. In the prostate, 1.6–1.9 times more T than DHT was required to achieve a half-maximal response for each of the three mark...

Apoptosis is the mode of keratinocyte death in cutaneous graft-versus-host disease

BACKGROUND Satellite cell necrosis is a histopathologic hallmark of cutaneous graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation. Although this interaction of lymphocytes with keratinocytes has features of immune cytolytic destruction, the details of the process are not known. OBJECTIVE Our purpose was to investigate whether apoptosis is involved in the process of GVHD. METHODS We used TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique with biotinylated nucleotides. Formalin-fixed, paraffin-embedded sections from skin biopsy specimens of patients with GVHD and normal skin were studied. RESULTS Labeling of scattered individual keratinocytes in the epidermis and adnexal epithelium undergoing cytolytic degeneration and a proportion of the adjacent lymphocytes was noted in all specimens of GVHD. CONCLUSION The positive staining of keratinocytes by the TUNEL method indicates that cell destruction in cutaneous GVHD involves apoptosis.

Insulin‐like growth factor binding protein 5 is associated with involution of the ventral prostate in castrated and finasteride‐treated rats

Insulin‐like growth factor binding protein (IGFBP)‐5 has been proposed as a signal for apoptosis in the ovary. To determine the relationship between IGFBP‐5 and apoptosis during regression of the androgen‐deprived prostate, rats were castrated or treated with the 5α‐reductase inhibitor finasteride for 4, 9, 14, 21, and 28 days.

Prostatic involution in men taking finasteride is associated with elevated levels of insulin-like growth factor-binding proteins (IGFBPs)-2, -4, and -5

Insulin‐like growth factor‐binding proteins (IGFBPs)‐2, ‐4, and ‐5 are associated with upregulation of apoptosis in the ovary. The purpose of this study was to assess the roles of IGF‐I and IGFBPs during involution of the prostate. Frozen and fixed tissue was collected by transurethral prostatectomy from Caucasian men, aged 52–82 years, scheduled for prostatectomy for benign prostatic hyperplasia, who took either placebo (n = 7) or the 5α‐reductase inhibitor finasteride for 6 days to 6 years (n = 15) prior to surgery.

Testosterone and prolactin increase carboxypeptidase‐D and nitric oxide levels to promote survival of prostate cancer cells

Plasma‐membrane carboxypeptidase‐D (CPD) releases arginine from extracellular substrates. Arginine is converted intracellularly to nitric oxide (NO). This study determined the effects of testosterone (T) and prolactin (PRL) on CPD expression, and the role(s) of CPD in NO production and survival of prostate cancer (PCa) cells.

The utility of tissue transglutaminase as a marker of apoptosis during treatment and progression of prostate cancer.

PURPOSE To determine the extent of cell proliferation and apoptosis during treatment and progression of prostate cancer and to determine whether staining for tissue transglutaminase is a better histological marker than TUNEL for neoadjuvant androgen ablation treatment of localized prostate cancer. MATERIALS AND METHODS Immunocytochemistry techniques were used on archival prostate tissue from four groups of men: 14 men with BPH, 18 men with untreated, localized prostate cancer, 21 men with localized prostate cancer who received neoadjuvant hormone therapy prior to prostatectomy and 18 men with metastatic androgen-independent prostate cancer. Cell proliferation was evaluated by staining for the Ki67 nuclear antigen, and apoptosis was evaluated by staining for DNA fragmentation (TUNEL technique) and tissue transglutaminase (tTG). Image analysis was used to quantitate the results. RESULTS TUNEL staining increased by 37% in localized prostate cancer compared with BPH, with a further increase of 43% seen after neoadjuvant therapy, although variation was such that neither was statistically significant. In androgen-independent cancer, TUNEL staining was decreased compared with neoadjuvant hormone treated cancer (p = 0.02). Staining for tTG was not increased in untreated prostate cancer compared with BPH; however, staining more than doubled after neoadjuvant therapy, compared with untreated prostate cancer (p = 0.04). Staining for tTG was markedly decreased in androgen-independent cancer (p = 0.07 compared with BPH and p = 0.0004 compared with neoadjuvant hormone treated cancer). Ki67 immunoreactivity did not significantly change in localized prostate cancer, either before or after neoadjuvant therapy, compared with BPH, but it more than doubled in androgen-independent prostate cancer (p = 0.07 compared with BPH and p = 0.05 compared with untreated prostate cancer). CONCLUSIONS This study shows that cell proliferation increases and apoptosis decreases as prostate cancer progresses to androgen independence, and, that of the markers used in this study, tissue transglutaminase most accurately reflects the anticipated effect of neoadjuvant hormone therapy on localized prostate cancer. An assessment of these parameters provides a valuable tool for appraising new prostate cancer therapies.

CAT-1-mediated arginine uptake and regulation of nitric oxide synthases for the survival of human breast cancer cell lines.

Growth of the human MCF‐7 breast cancer cell line is highly dependent on L‐arginine. We have reported that L‐arginine, released from extracellular substrates by prolactin (PRL)‐ and 17β‐estradiol (E2)‐induced carboxypeptidase‐D in the cell membrane, promotes nitric oxide (NO) production for MCF‐7 cell survival. Arginine uptake is mediated by members of the cationic amino acid transporter (CAT) family and may coincide with induction of nitric oxide synthase (NOS) for the production of NO. The present study investigated the CAT isoforms and PRL/E2 regulation of CAT and NOS in breast cancer cell lines. Using RT‐PCR analysis, CAT‐1, CAT‐2A, and CAT‐2B transcripts were detected in MCF‐7, T47D, and MDA‐MB‐231 cells. The CAT‐4 transcript was detected in MDA‐MB‐231 only. CAT‐3 was not detected in any of these cells. PRL and E2 did not significantly alter levels of CAT‐1 mRNA and protein, nor CAT‐2A and CAT‐2B mRNAs in MCF‐7 and T47D cells. PRL and E2 also had no effect on the overall uptake of L‐[2,3,4,5‐H3] arginine into these cells. However, confocal immunofluorescent microscopy showed that PRL and E2 upregulated eNOS and iNOS proteins, which distributed in the cytoplasm and/or nucleus of MCF‐7 cells. Knockdown of CAT‐1 gene expression using small interfering RNA significantly decreased L‐[2,3,4,5‐H3]‐arginine uptake, decreased viability and increased apoptosis of MCF‐7 and T47D cells. In summary, several CAT isoforms are expressed in breast cancer cells. The CAT‐1 isoform plays a role in arginine uptake and, together with PRL/E2‐induced NOS, contribute to NO production for the survival of MCF‐7 and T47D cells. J. Cell. Biochem. 112: 1084–1092, 2011.

Overexpression of 5 alpha-Reductase Type 1 Increases Sensitivity of Prostate Cancer Cells to Low Concentrations of Testosterone

Conversion of testosterone to dihydrotestosterone (DHT) by the enzymes 5α‐reductase types 1 (5αR1) and 2 (5αR2) is important for normal and pathological growth of the prostate. The predominant isoenzyme in normal prostate is 5αR2. However, prostate cancer (PCa) development is accompanied by a decrease in 5αR2 and an increase in 5αR1. The biological significance of increased 5αR1 expression is not fully understood. Therefore, the aim of this study was to determine the effect of overexpression of 5αR1 on growth and prostate‐specific antigen (PSA) production in PCa cells.

Carboxypeptidase-D is elevated in prostate cancer and its anti-apoptotic activity is abolished by combined androgen and prolactin receptor targeting

Carboxypeptidase‐D (CPD) cleaves C‐terminal arginine for nitric oxide (NO) production. CPD and NO levels are upregulated by testosterone (T) and prolactin (PRL) to promote survival of prostate cancer (pCa) cells. This study evaluated CPD immunostaining and T/PRL regulation of CPD and NO levels in benign and malignant prostate tissues/cells to determine the role of CPD in pCa.

Progestin-inducible EDD E3 ubiquitin ligase binds to α4 phosphoprotein to regulate ubiquitination and degradation of protein phosphatase PP2Ac

Mammalian α4 phosphoprotein binds to the protein phosphatase 2A catalytic subunit (PP2Ac) to regulate PP2A activity, and to poly(A)-binding protein (PABP) and progestin-inducible EDD E3 ubiquitin ligase. This study showed induction of the EDD protein by progesterone, 17β-estradiol and prolactin in breast cancer cells. Co-immunoprecipitation analyses, using lysates of COS-1 cells transfected with α4-deletion constructs, showed the α4 N-terminus binding to endogenous PP2Ac and PABP, and the C-terminus to EDD. Monoubiquitinated α4 in MCF-7 cells was unaffected by EDD-targeting siRNA (siEDD) nor by non-targetting siNT, thus, EDD does not ubiquitinate α4. PP2Ac is polyubiquitinated, and 36-kDa PP2Ac only was detected in siEDD- or siNT-transfected cells. However, treatment with proteasomal inhibitor MG132 showed polyubiquitinated-PP2Ac molecules (∼65-250kDa) abundantly in siNT controls but low in siEDD-transfectants, implicating PP2Ac as an EDD substrate. Finally, progesterone induction of EDD in MCF-7 cells correlated with decreased PP2Ac levels, further implicating hormone-inducible EDD in PP2Ac turnover.