It's not my fault, blame it on my microenvironment.
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Animals↗
Biomedical subjects
Gerald R Cunha
Publications and source records attributed to Gerald R Cunha.
Role of stroma in carcinogenesis of the prostate.
Prostatic development is induced by androgens acting via mesenchymal-epithelial interactions. Androgens elicit their morphogenetic effects by acting through androgen receptors (ARs) in urogenital sinus mesenchyme (UGM), which induces prostatic epithelial development. In adulthood reciprocal homeostatic stromal-epithelial interactions maintain functional differentiation and growth-quiescence. Testosterone plus estradiol (T+E2) have been shown to induce prostatic carcinogenesis in animal models. Thus, tissue recombinant studies were undertaken to explore the mechanisms of prostatic carcinogenesis in BPH-1 cells in which ARs and estrogen receptors (ERs) are undetectable. For this purpose, BPH-1 cells were combined with UGM, and the UGM+BPH-1 recombinants were grafted to adult male hosts. Solid branched epithelial cords and ductal structures formed in untreated UGM+BPH-1 recombinants. Growth was modest, and tumors did not develop. UGM+BPH-1 recombinants treated with T+E2 formed invasive carcinomas. BPH-1 cells lack ARs and ERs, whereas rat UGM expresses both of these receptors. These data show that immortalized nontumorigenic human prostatic epithelial cells can undergo hormonal carcinogenesis in response to T+E2 stimulation via paracrine mechanisms and demonstrate that the stromal environment plays an important role in mediating hormonal carcinogenesis. During prostatic carcinogenesis the stroma undergoes progressive loss of smooth muscle with the appearance of carcinoma-associated fibroblasts (CAF). This altered stroma was tested for its ability to promote carcinogenesis of nontumorigenic but immortalized human prostatic epithelial cells (BPH-1). CAF+BPH-1 tissue recombinants formed large carcinomas. In contrast, recombinants composed of normal prostatic stroma+BPH-1 cells exhibited minimal growth. This stroma-induced malignant transformation was associated with additional genetic alterations and changes in gene expression. Thus, alteration in the stromal microenvironment was sufficient to promote malignant transformation of human prostatic epithelial cells.
Androgens↗
Stable maintenance of 5alpha-reductase activity in long-term subcultures of fibroblasts derived from the foreskin.
BACKGROUND: There is up to a 50-fold variation in control subjects in current assays of 5alpha-reductase activity which makes interpretation difficult. It was therefore attempted in this study to establish an assay method which produced stable 5alpha-reductase activity in long-term subcultured foreskin fibroblasts. METHODS: Foreskin fibroblasts were obtained from three boys with phimosis (control subjects), three patients with Reifenstein syndrome and one patient with 5alpha-reductase deficiency (due to mutation L113P in exon 2 of the SRD5A2 gene). To maximize the number of cells in the DNA synthesis phase, cells were subcultured consistently to approximately 70% confluency. Thawed cells, frozen after the third subculture, were incubated for 24 h with [1beta,2beta-3H] testosterone. 5alpha-Reductase activity was expressed as the sum of formed [3H] 5alpha-reduced metabolites (separated by thin-layer chromatography). RESULTS: The full range of 5alpha-reductase activity in controls and patients with Reifenstein syndrome was 3.44-15.59 pmol/h per mg protein: a 4.53-fold variation. The activity in the patient with 5alpha- reductase deficiency was 0.52 pmol/h per mg protein. CONCLUSION: By the cell culture methods used in this study, which aimed to increase the number of cells in the DNA synthesis phase, foreskin fibroblasts maintained a considerably stable level of 5alpha-reductase activity during long-term subculture. Therefore, this assay method can be used for differential diagnosis of 5alpha-reductase deficiency from other relevant entities.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
A novel method for growing human breast epithelium in vivo using mouse and human mammary fibroblasts.
A novel system is described for studying the growth of normal human mammary epithelium in vivo as grafts in athymic nude mice. The key feature of this model is reconstitution of the epithelial-stromal interactions required for normal growth and differentiation of the human mammary epithelium, which produces ducts that are comparable to those in the normal human mammary gland. Human breast epithelial organoids were combined with mammary fibroblasts from mouse or human origin in collagen gels, which were subsequently transplanted under the renal capsule of female nude mice hosts. The resulting grafts showed an increase in the ductal density compared with that observed previously. These ducts expressed appropriate markers for luminal and myoepithelial cells and steroid receptors. Treatment of the host with diethylstilbestrol or estradiol and progesterone significantly increased the number of ducts observed and increased cell proliferation. The grafts also displayed production of beta-casein and milk fat globule membrane protein when the hosts were allowed to become pregnant. This model allows for a variety of epithelial and stromal cells to be used in combination, which would aid in understanding key factors that regulate normal human mammary gland development.
Animals↗
Role of systemic and local IGF-I in the effects of estrogen on growth and epithelial proliferation of mouse uterus.
IGF-I is a critical regulator of uterine growth, and locally produced uterine IGF-I could mediate the effects of 17beta-E2 on growth and cellular proliferation. We used IGF-I knockout (KO) mice and tissue grafting to determine the roles of local and systemic IGF-I in uterine growth and E2 responsiveness. Uteri from adult KO mice and neonatal and adult wild-type (WT) mice were grown under the renal capsule of female athymic mice for 4 wk. Initial uterine weights of adult KO and neonatal WT mice were 5% or less of those of adult WT uteri. Weights of adult WT uterine grafts did not increase during grafting. Weights of adult KO and neonatal WT uteri exposed to normal systemic levels of IGF-I in athymic hosts increased 20- to 30-fold to equal or exceed those of adult WT grafts. Uterine epithelial height, reduced in KO mice, was restored to WT levels in KO uteri grafted into athymic hosts. The absence of local IGF-I production in KO uteri did not impair E2- induced epithelial proliferation in KO uterine grafts. Neonatal WT uteri grafted into KO hosts showed minimal growth, providing evidence that local uterine IGF-I production is insufficient to support uterine growth in the absence of systemic IGF-I. E2 treatment of KO females produced minimal uterine growth, confirming that lack of IGF-I, rather than E2, caused the uterine hypoplasia. In summary, systemic IGF-I supports normal uterine growth and E2 response in the absence of local IGF-I. Local IGF-I is not a direct mediator of E2 action in uterus, and systemic IGF-I may be more important than previously thought for growth of the uterus and other tissues.
Animals↗
The role of smooth muscle in regulating prostatic induction.
We have examined the role that smooth muscle plays during prostatic organogenesis and propose that differentiation of a smooth muscle layer regulates prostatic induction by controlling mesenchymal/epithelial interactions. During development of the rat reproductive tract, an area of condensed mesenchyme involved in prostatic organogenesis is formed. This mesenchyme (the ventral mesenchymal pad, VMP) is found in both males and females, yet only males develop a prostate. We demonstrate that a layer of smooth muscle differentiates between the VMP and the urethral epithelium, and that there is a sexually dimorphic difference in the development of this layer. Serial section reconstruction showed that the layer formed at approximately embryonic day 20.5 in females, but did not form in males. In cultures of female reproductive tracts, testosterone was able to regulate the thickness of this layer resulting in a 2.4-fold reduction in thickness. We observed that prostatic buds were present in some female reproductive tracts, and determined that testosterone was able to stimulate prostatic organogenesis, depending upon the bud position relative to the smooth muscle layer. In vitro recombination experiments demonstrated that direct contact with the VMP led to the induction of very few epithelial buds, and that androgens dramatically increased bud development. Taken together, our data suggest that differentiation of a smooth muscle layer regulates signalling between mesenchyme and epithelium, and comprises part of the mechanism regulating prostatic induction.
Animals↗
Growth, regeneration, and tumorigenesis of the prostate activates the PSCA promoter.
The prostate gland undergoes dramatic changes in growth status during normal physiologic development, following androgen administration to castrate animals, and during tumor development. The prostate stem cell antigen (PSCA, named for its strong sequence homology to the thymocyte marker stem cell antigen 2) is a cell surface molecule associated with human and murine prostate cancer. To help define the regulation of this molecule, we created a transgenic mouse strain, which uses the human PSCA promoter region to control the expression of enhanced green fluorescent protein (GFP). Expression of GFP was detected in mid-gestation following the appearance of prostatic buds from the urogenital sinus. In adult mice, GFP expression was restricted to a subset of cells located in the distal tips of the glands. GFP expression increased during puberty and regeneration driven by androgen and associated with expansive growth of the prostate. GFP-positive cells coexpressed markers associated with both basal and secretory cells in the human prostate. Prostate carcinogenesis driven by T antigen in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model results in an increased percentage and intensity level for PSCA promoter-driven GFP-positive cells. This transgenic system helps define the range of cellular changes associated with altered expression of PSCA, shows that transcriptional control is a major component regulating PSCA levels, and provides a useful tool to study subpopulations of prostate epithelial cells and factors that regulate the PSCA promoter.
Animals↗
Expression of the androgen receptor and 5 alpha-reductase type 2 in the developing human fetal penis and urethra.
Normal penile development is dependent on testosterone, its conversion via steroid 5 alpha-reductase type 2 to dihydrotestosterone, and a functional androgen receptor (AR). The goal of this study was to investigate the distribution of AR and 5 alpha-reductase type 2 in the developing human fetal external genitalia with special emphasis on urethra formation. Twenty fetal genital specimens from normal human males (12-20 weeks gestation) were sectioned serially and stained by avidin-biotinylated peroxidase complex method with antigen retrieval. Stained sections throughout male genital development documented the expression of AR and 5 alpha-reductase type 2 in the phallus. Between 12 and 14 weeks of gestation, AR was localized to epithelial cells of the urethral plate in the glans, the tubular urethra of the penile shaft, and stromal tissue surrounding the urethral epithelium. In the fetal penis between 16 and 20 weeks gestation, the density of AR expression was greatest in urethral epithelial cells versus the surrounding stromal tissues. There was a characteristic pattern of AR expression in the glandular urethral epithelium between 16 and 20 weeks gestation. AR expression was greater along the ventral aspect of the glandular urethra than along the dorsal aspect of the urethral epithelium. The expression of 5 alpha-reductase type 2 was localized to the stroma surrounding the urethra, especially along the urethral seam area in the ventral portion of the remodeling urethra. These anatomical studies support the hypothesis that androgens are essential for the formation of the ventral portion of the urethra and that abnormalities in either the AR or 5 alpha-reductase type 2 can explain the occurrence of hypospadias.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗