The uterine epithelial loss of Pten is inefficient to induce endometrial cancer with intact stromal Pten.

Mutation of the tumor suppressor Pten often leads to tumorigenesis in various organs including the uterus. We previously showed that Pten deletion in the mouse uterus using a Pgr-Cre driver (Ptenf/fPgrCre/+) results in rapid development of endometrial carcinoma (EMC) with full penetration. We also reported that Pten deletion in the stroma and myometrium using Amhr2-Cre failed to initiate EMC. Since the Ptenf/fPgrCre/+ uterine epithelium was primarily affected by tumorigenesis despite its loss in both the epithelium and stroma, we wanted to know if Pten deletion in epithelia alone will induce tumorigenesis. We found that mice with uterine epithelial loss of Pten under a Ltf-iCre driver (Ptenf/f/LtfCre/+) develop uterine complex atypical hyperplasia (CAH), but rarely EMC even at 6 months of age. We observed that Ptenf/fPgrCre/+ uteri exhibit a unique population of cytokeratin 5 (CK5) and transformation related protein 63 (p63)-positive epithelial cells; these cells mark stratified epithelia and squamous differentiation. In contrast, Ptenf/fLtfCre/+ hyperplastic epithelia do not undergo stratification, but extensive epithelial cell apoptosis. This increased apoptosis is associated with elevation of TGFß levels and activation of downstream effectors, SMAD2/3 in the uterine stroma. Our results suggest that stromal PTEN via TGFß signaling restrains epithelial cell transformation from hyperplasia to carcinoma. In conclusion, this study, using tissue-specific deletion of Pten, highlights the epithelial-mesenchymal cross-talk in the genesis of endometrial carcinoma.

Distinct properties of cyclin-dependent kinase complexes containing cyclin A1 and cyclin A2.

The distinct expression patterns of the two A-type cyclins during spermatogenesis and the absolute requirement for cyclin A1 in this biological process in vivo suggest that they may confer distinct biochemical properties to their CDK partners. We therefore compared human cyclin A1- and cyclin A2-containing CDK complexes in vitro by determining kinetic constants and by examining the complexes for their ability to phosphorylate pRb and p53. Differences in biochemical activity were observed in CDK2 but not CDK1 when complexed with cyclin A1 versus cyclin A2. Further, CDK1/cyclin A1 is a better kinase complex for phosphorylating potentially physiologically relevant substrates pRb and p53 than CDK2/cyclin A2. The activity of CDKs can therefore be regulated depending upon which A-type cyclin they bind and CDK1/cyclin A1 might be preferred in vivo.

Conditional loss of uterine Pten unfailingly and rapidly induces endometrial cancer in mice.

Etiology of endometrial cancer (EMC) is not fully understood. Animal models with rapidly and spontaneously developing EMC will help explore mechanisms of cancer initiation and progression. Pten(+/-) mice are currently being used as a model to study EMC. These females develop atypical endometrial hyperplasia of which approximately 20% progresses to EMC. In addition, tumors develop in other organs, complicating the use of this model to specifically study EMC. Here, we show that conditional deletion of endometrial Pten results in EMC in all female mice as early as age 1 month with myometrial invasion occurring by 3 months. In contrast, conditional deletion of endometrial p53 had no phenotype within this time frame. Whereas mice with endometrial Pten deletion had a life span of approximately 5 months, mice with combined deletion of endometrial Pten and p53 had a shorter life span with an exacerbated disease state. Such rapid development of EMC from homozygous loss of endometrial Pten suggests that this organ is very sensitive to this tumor suppressor gene for tumor development. All lesions at early stages exhibited elevated Cox-2 and phospho-Akt levels, hallmarks of solid tumors. More interestingly, levels of two microRNAs miR-199a(*) and miR-101a that posttranscriptionally inhibit Cox-2 expression were down-regulated in tumors in parallel with Cox-2 up-regulation. This mouse model in which the loxP-Cre system has been used to delete endometrial Pten and/or p53 allows us to study in detail the initiation and progression of EMC. These mouse models have the added advantage because they mimic several features of human EMC.

Insights into male germ cell apoptosis due to depletion of gonadotropins caused by GnRH antagonists.

The role of pituitary gonadotropins in the regulation of spermatogenesis has been unequivocally demonstrated, although, the precise mechanism of this regulation is not clearly understood. Previous studies have shown that specific immunoneutralization of LH/testosterone caused apoptotic cell death of meiotic and post-meiotic germ cells while that of FSH resulted in similar death of meiotic cells. In the present study, the death process of germ cells has been characterized by depleting both FSH and testosterone by administering two different potent GnRH antagonists, Cetrorelix and Acyline to both rats and mice. Pro-survival factors like Bcl-2 and Bcl-x/l were unaltered in germ cells due to GnRH antagonist treatment, although a significant increase in several pro-apoptotic markers including Fas and Bax were evident at both protein and RNA levels. This culminated in cytochrome C release from mitochondria and eventually increase in the activity of caspase-8 and caspase-3. These data suggest that both extrinsic and intrinsic apoptotic death pathways are operative in the germ cells death following decrease in FSH and testosterone levels. Multiple injections of GnRH antagonist resulted in complete disappearance of germ cells except the spermatogonial cells and discontinuation of the treatment resulted in full recovery of spermatogenesis. In conclusion our present data suggest that the principal role of FSH and testosterone is to maintain spermatogenic homeostasis by inhibiting death signals for the germ cells.

Expression of apoptotic genes in testicular germ cells and their modulation by luteinizing hormone.

Gonadotropins regulate spermatogenesis by promoting survival and differentiation of germ cells. The molecular markers that are modulated by these hormones to ensure survival however have not been described in great detail. Immunoneutralization of LH in particular leads to apoptotic cell death of the spermatocytes and the round spermatids. In the present study, the expression pattern and regulation of apoptotic markers after specific immunoneutralization of LH in germ cells purified from rats has been investigated at the RNA and protein level. Of the several markers tested, Bax, caspases 1 and 2 and Fadd exhibit differential expression, with the round spermatids expressing higher levels of caspases 1 and 2, and the spermatocytes expressing higher levels of Bax and Fadd. The two cell types therefore exhibit differential expression of apoptotic markers. The cell types also differ with respect to their response to LH antiserum treatment. Fas and Bax both are up-regulated in the round spermatids after 24h of antiserum treatment. In the spermatocytes, Fas was up-regulated as early as 12h after antiserum treatment while Bax was up-regulated after 2 days. These results demonstrate that LH regulates survival of germ cells by modulating the levels of pro and anti-apoptotic proteins.