Eeva Valve

Vascular endothelial growth factors are differentially regulated by steroid hormones and antiestrogens in breast cancer cells

Vascular endothelial growth factor (VEGF) is a major inducer of tumor angiogenesis and an important prognostic factor in breast cancer. Hypoxia is an important inducer of VEGF expression but less is known of the role of hormones in VEGF regulation. We have studied the regulation of VEGF, VEGF-B, VEGF-C, and VEGF-D mRNAs in human MCF-7 and mouse S115 breast carcinoma cells stimulated by estrogens and androgens, respectively. VEGF, VEGF-B, and VEGF-C were expressed in both cell lines, whereas VEGF-D was expressed only in S115 cells. Addition of estradiol (E2) caused a biphasic increase of VEGF mRNA in MCF-7 cells and led to accumulation of the VEGF protein in the culture medium. The VEGF-B mRNA was not affected, while a decrease occurred in VEGF-C mRNA. Similarly, testosterone upregulated the expression of VEGF mRNA in the S115 cells. Experiments with actinomycin D and cycloheximide suggested that estrogen induction of VEGF mRNA is dependent on the synthesis of new mRNA and increased mRNA half-life. The antiestrogen ICI 182.780 inhibited E2 stimulation of VEGF, suggesting that the effect was mediated by the estrogen receptor. In contrast, the antiestrogens tamoxifen and toremifene which inhibit MCF-7 cell growth in vivo and in vitro did not inhibit estrogen effect but induced VEGF mRNA expression when used alone. The antiandrogen cyprosterone acetate inhibited T induction of VEGF mRNA in S115 cells, thus suggesting that activation of androgen receptor must be involved in the increase of VEGF mRNA. Our results suggest that both estrogen and androgen stimulate the expression of VEGF by increasing gene transcription and mRNA stability. In addition, the antiestrogens tamoxifen and toremifene also increased VEGF expression. Estrogen and androgen induction of VEGF expression and promotion of new vessel formation may be an important paracrine mechanism by which these hormones contribute to the early phase of tumor growth of hormonal cancer.

Prolactin and prolactin receptors are expressed and functioning in human prostate.

Prolactin is widely expressed in different tissues, and it is presumed to have both local and systemic actions. In males it is known to influence reproductive functions but the significance and mechanisms of prolactin action in male accessory reproductive tissues are poorly understood. Here we show that prolactin acts as a direct growth and differentiation factor for human prostate, as measured by changes in DNA synthesis and epithelial morphology of organ cultures. Furthermore, we report the expression in human prostate of a short prolactin receptor form in addition to the long form, based upon ligand cross-linking studies and RT-PCR analysis of mRNA expression. The highest density of prolactin receptors was detected in the secretory epithelial cells by immunohistochemistry. Finally, we report that prolactin is locally produced in human prostate epithelium, as evidenced by marked prolactin immunoreactivity in a significant portion of prostate epithelial cells, with parallel expression of prolactin mRNA in human prostate. Collectively, these data provide significant support for the existence of an autocrine/paracrine loop of prolactin in the human prostate and may shed new light on the involvement of prolactin in the etiology and progression of neoplastic growth of the prostate.

Increased expression of cyclooxygenase-2 and nitric oxide synthase-2 in human prostate cancer

Abstract Cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase-2 (NOS-2) each have an important role in angiogenesis. The expression of these genes was investigated in human prostate cancer by immunohistochemistry, the expression of COX-1 and COX-2 being confirmed by mRNA analysis. Prostate cancer specimens from 12 patients were compared to control prostates from 13 patients operated on for bladder carcinoma. The intensity of COX-2 and NOS-2 immunostaining was significantly stronger in prostate cancer cells than in the non-malignant glandular epithelium of the control prostates. COX-2 and NOS-2 were clearly also expressed in the lesions of prostatic intraepithelial neoplasia (PIN) in control prostates. COX-2 was detected in the muscle fibres of the hyperplastic stroma of some control prostates. No significant difference was detected in COX-1 expression between control and cancer prostates. These results indicate that the expression of COX-2 and NOS-2 is elevated in prostatic adenocarcinoma and in PIN.

Differential expression of estrogen receptors α and β in adult rat accessory sex glands and lower urinary tract

Abstract Estrogens induce pronounced structural and functional changes in male accessory sex glands and the lower urinary tract in both sexes, but the exact mechanisms of estrogen action are not fully understood. This study was undertaken to localise the tissue cell types that express estrogen receptor in adult rats, and to determine the receptor subtype (ERα and ERβ) in order to identify sites that may respond directly to estrogens. In the male accessory sex glands (seminal vesicles, prostatic lobes and ampullary glands), ERβ mRNA and protein were strongly expressed in the epithelium but not in the stroma, while ERα mRNA was present only in the fibromuscular tissue surrounding the prostatic collecting ducts in the posterior periurethral region and in ampullary gland stroma. In the epithelium of the urinary bladder and urethra of both sexes, high level of ERβ mRNA and protein, but no ERα mRNA, was detected. The connective tissue in urinary bladder of both males and females, as well as that in prostatic urethra in males expressed ERα mRNA. The neural cells in the autonomic ganglia of the prostatic plexus were strongly positive for ERβ mRNA, but were completely devoid of ERα. We conclude that ERβ is the predominant ER subtype in the epithelium of adult male rat accessory sex glands and the lower urinary tract of both males and females, as well as in the prostatic neural plexus regulating the function of the lower urinary tract in males, while ERα is present only in the stromal compartment of distinct sites. These results indicate that in these tissues in intact adults there are multiple targets for direct estrogen action. Furthermore, the differential or complementary expression of the two ER subtypes suggests that they may have specific functions, and may explain the complex structural and functional changes induced by estrogens.

Androgen-dependent expression of prolactin in rat prostate epithelium in vivo and in organ culture.

Peptide hormones and growth factors are involved in the regulation of prostatic cell proliferation, differentiation, and programmed cell death, which functions are primarily controlled by androgen. In carcinogenesis, prostatic cancer cells often lose androgen dependence and become largely dependent on local growth factors. The prostatic cancer cells able to respond to factors other than androgen by proliferation and inhibition of apoptosis are possibly able to survive. We demonstrate that prostatic epithelium expresses prolactin mRNA and protein in a characteristic manner. By using in situ hybridization, an overall distribution of prolactin mRNA was demonstrated in the epithelium of rat dorsal and lateral prostate, whereas a very specific localization of prolactin protein to single cells was observed by immunohistochemistry in the same tissues. In these cells, immunoelectron microscopy showed that prolactin was primarily localized to the secretory granules. These data demonstrate a selective regulation of prostatic prolactin at least at the level of transcript processing/translation and/or protein accumulation and secretion. In addition, the expression of prolactin protein in rat dorsal and lateral prostate was found to be androgen dependent in vivo in castrated and in castrated, testosterone‐treated rats, as well as in vitro in organ cultures. Our results support the concept of an autocrine/paracrine loop of prolactin action in prostate where it could mediate some of androgen actions. Also, locally synthesized prolactin might belong to the factors that take over androgen regulation of prostatic cancer cells during the development of androgen‐independent growth.—Nevalainen, M. T., Valve, E. M., Ahonen, T., Yagi, A., Paranko, J., Härkönen, P. L. Androgen‐dependent expression of prolactin in rat prostate epithelium in vivo and in organ culture. FASEB J. 11, 1297–1307 (1997)

Erratum to “Differential expression of estrogen receptors α and β in adult rat accessory sex glands and lower urinary tract”[Mol. Cell. Endocrinol. 164 (2000) 109–116]

Estrogens induce pronounced structural and functional changes in male accessory sex glands and the lower urinary tract in both sexes, but the exact mechanisms of estrogen action are not fully understood. This study was undertaken to localise the tissue cell types that express estrogen receptor in adult rats, and to determine the receptor subtype (ERa and ERb) in order to identify sites that may respond directly to estrogens. In the male accessory sex glands (seminal vesicles, prostatic lobes and ampullary glands), ERb mRNA and protein were strongly expressed in the epithelium but not in the stroma, while ERa mRNA was present only in the fibromuscular tissue surrounding the prostatic collecting ducts in the posterior periurethral region and in ampullary gland stroma. In the epithelium of the urinary bladder and urethra of both sexes, high level of ERb mRNA and protein, but no ERa mRNA, was detected. The connective tissue in urinary bladder of both males and females, as well as that in prostatic urethra in males expressed ERa mRNA. The neural cells in the autonomic ganglia of the prostatic plexus were strongly positive for ERb mRNA, but were completely devoid of ERa. We conclude that ERb is the predominant ER subtype in the epithelium of adult male rat accessory sex glands and the lower urinary tract of both males and females, as well as in the prostatic neural plexus regulating the function of the lower urinary tract in males, while ERa is present only in the stromal compartment of distinct sites. These results indicate that in these tissues in intact adults there are multiple targets for direct estrogen action. Furthermore, the differential or complementary expression of the two ER subtypes suggests that they may have specific functions, and may explain the complex structural and functional changes induced by estrogens.

Prostatic dysplasia associated with increased expression of C-MYC in neonatally estrogenized mice

Neonatal estrogenization of the mouse with diethylstilbestrol (DES; 2 micrograms./pup/day for days 1 to 3) or 17 beta-estradiol (200 micrograms./pup/day for days 1 to 3) resulted in epithelial dysplasia in the posterior periurethral region of the prostate at the age of 1 year. The dysplastic lesions ranged from mild to severe and, in addition to emergence of nuclear anaplasia, the architectural pattern of the glands was disturbed. Prenatal estrogenization (100 micrograms./kg. of maternal body weight on days 13 and 15 of gestation) only resulted in mild epithelial hyperplasia and occasional dysplasia in the ventral lobe of the prostate, but not in the posterior periurethral region. When neonatally estrogenized mice were allowed to grow until the age of 18 months, the degree and extent of the dysplasia of the posterior periurethral region was increased, but no frank invasion or metastases could be demonstrated. Combined estrogen and androgen treatment of neonatally estrogenized mice for 3 months (between 9 and 12 months of age) augmented nuclear dysplasia, but no invasive growth was seen in this group, either. Mild epithelial dysplasia was found in the dorsolateral lobes and coagulating glands of similarly treated control animals. A relation between the activation of certain proto-oncogenes and the development of several cancers has been shown in humans and experimental animals. In the present study, Northern blot analysis of total RNAs showed that the levels of c-myc mRNA were increased in the ventral and dorsolateral lobes, coagulating glands and prostatic urethra of neoDES mice at the age of 9 months. However, it remains to be determined whether the increase in c-myc expression is involved in the development of hyperplastic and dysplastic changes in the prostate of neoDES mice.

Increased Expression of FGF-8 Isoforms and FGF Receptors in Human Premalignant Prostatic Intraepithelial Neoplasia Lesions and Prostate Cancer

Fibroblast growth factor 8 (FGF-8) is implicated in growth of prostate cancer. Alternative splicing of the human FGF-8 gene potentially allows coding for four protein isoforms (a, b, e, and f). These isoforms differ in their binding to FGF receptors (FGFR) and in their mitogenic and transforming capacity in transfection assays. Here, we used RT-PCR and immunohistochemistry to study the expression of FGF-8 and FGFR isoforms in human prostate cancer (n = 31). Nonmalignant prostate specimens from cystoprostatectomies (n = 24) were examined as controls. Most prostate cancer samples and some control prostates also contained prostatic intraepithelial neoplasia (PIN) lesions. FGF-8a and e were expressed at significantly higher frequencies in prostate cancer (FGF-8a, 55%; FGF-8e, 45%) than in control samples (FGF-8a, 17%, p = 0.0052; FGF-8e, 8%, p = 0.0031). On the contrary, FGF-8b was found at an equal frequency in prostate cancer (55%) and in control prostates (50%). Furthermore, a combination of two or three FGF-8 isoforms (a, b, and/or e) was also expressed at a higher frequency in prostate cancer than in control samples (45% and 8%, respectively, p = 0.0031). Immunohistochemistry with an antibody recognizing all FGF-8 isoforms was more strongly immunoreactive in prostate cancer cells and PIN lesions than in normal-type epithelium. The receptor splicing variants FGFR1IIIc and FGFR2IIIc, which are activated by FGF-8, were found both in prostate cancer and control samples. Interestingly, immunoreactivity for FGFR1 and FGFR2 was much stronger in prostate cancer cells and PIN than in normal epithelium. These results demonstrate, for the first time, that FGF-8 isoforms and their receptors FGFR1IIIc and FGFR2IIIc are expressed at an increased level not only in prostate cancer but also in premalignant PIN lesions. These data suggest that FGF-8 may have an important autocrine role in the development of human prostate cancer. In addition to FGF-8b, the FGF-8 isoforms a and e may be involved in this process.

Expression of fibroblast growth factor (FGF)-8 isoforms and FGF receptors in human ovarian tumors

FGF‐8 is a mitogenic growth factor, which is widely expressed during embryonic development but only at a very low level in adult tissues. Alternative splicing of the human FGF‐8 gene potentially allows coding for 4 protein isoforms (a, b, e, f), which differ in their transforming capacity. The FGF‐8 isoforms preferentially activate the receptors FGFR1IIIc, FGFR2IIIc, FGFR3IIIc and FGFR4. FGF‐8 is over‐expressed in human breast and prostate cancers. Expression has also been found in RT‐PCR studies of human ovarian and testicular cancers. The present study was undertaken to examine which FGF‐8 isoforms are expressed in ovarian cancer and whether FGF‐8 receptors are also expressed. Specimens from 5 normal human ovaries and 51 ovarian tumors (1 benign tumor, 8 borderline malignancies, 42 malignant tumors of different histopathological types) were studied by RT‐PCR and immunohistochemistry. FGF‐8 isoform b was expressed in all ovarian tumors and in all 7 ovarian‐cancer cell lines studied. Isoform a was co‐expressed in 9 malignant ovarian tumors. FGF‐8 mRNA was not detected by RT‐PCR of 3 normal ovary samples. Immunohistochemical staining localized FGF‐8 protein to cancer cells. In general, the increased intensity of FGF‐8 staining was associated with loss of differentiation within the tumors (Bowkers test, p = 0.37). FGF‐8 staining of surface epithelium observed on 2 normal ovaries was very faint. RT‐PCR showed that FGFR1IIIc, FGFR2IIIc and FGFR4 were the FGF‐8 receptors expressed in normal ovaries and in ovarian tumors. FGF‐8 receptor immunoreactivity was preferentially found in normal ovary surface epithelium and tumor cells but also in some stromal cells. Collectively, our results show that ovarian cancers of a wide variety of histological types expressing receptors for FGF‐8 have acquired the capacity of expressing FGF‐8. This suggests that FGF‐8 has an important role in ovarian tumorigenesis. Int. J. Cancer 88:718–725, 2000.

Calcitonin promotes osteoclast survival in vitro

Inactivation of resorbing osteoclasts by calcitonin is associated with typical morphological changes and alteration of the specific organization of osteoclast cytoskeleton. Here we show that calcitonin also promotes the survival of rat osteoclasts in vitro, cultured either on glass or bone, by delaying the onset of apoptosis. Parathyroid hormone had no effect on osteoclasts cultured on glass but it slightly increased apoptosis index of osteoclasts cultured on bone. Calcitonin was also able to rescue osteoclasts in calvarial explant cultures. The survival effect of calcitonin was mimicked by dibutyryl cAMP and could not be blocked by various metabolic inhibitors known to affect the apoptotic pathway. However, clodronate-induced apoptosis of osteoclasts could not be reversed by calcitonin and neither could calcitonin rescue osteoclasts already committed to apoptosis. It did not alter the distribution of Bcl-2 in osteoclasts. Our results show that at least in vitro calcitonin protects osteoclasts from apoptosis and suggest that it regulates the onset of apoptosis.