Category Archives: Human Trophoblast

Expression of Gt Cyclins: DISCUSSION(5)

DISCUSSION(5)

Overexpression of E2F1 in myoblasts inhibited expression of differentiation-dependent genes; however, the actions of a hyperactive pRb mutant could override these effects, demonstrating regulation of differentiation via cyclin/Cdk inactivation of pRb. Our results demonstrate that differentiation of cytotrophoblast into syncytiotrophoblast is associated with a decrease in the degree of phosphorylation of pRb resulting in predominantly hypophosphorylated, active pRb (Fig. 4c). Since cyclin E gene expression is regulated by E2F, sequestration of E2F by active, hypophosphorylated pRb might explain, in part, the observed down-regulation of cyclin E gene expression in this model system and may, in turn, serve to potentiate the differentiation process. Buy Asthma Inhalers Online

Expression of Gt Cyclins: DISCUSSION(4)

Immunohistochemical analysis limited cyclin D1 im-munoreactivity to intravillous blood vessels and extravil-lous cytotrophoblasts, but D1 immunoreactivity was not expressed in villous trophoblast in second- and third-trimester placentae. Cyclin D2 protein was expressed in proliferating ED27 cells and to a lesser extent in freshly isolated cytotrophoblast (Fig. 3) but was not detected by immunohistochemical analyses. It is likely that differences in the sensitivity of techniques could account for the discrepancies between these two studies.

Expression of Gt Cyclins: DISCUSSION(3)

DISCUSSION(3)

In most cell lines, p27KlP1 expression is primarily under posttranslational control, as mRNA levels typically remain constant while protein levels decrease throughout G, phase due to an increase in the rate of ubiquitin-targeted proteolytic breakdown. However, during terminal differentiation of trophoblasts, p27Kipl mRNA expression was up-regulated (Fig. 6b) comparable to the up-regulation described in cultured embryonic stem cells induced to form embryoid bodies. The increased expression of p27Kipl protein in cultured trophoblasts may reflect increases in mRNA (Fig. 6b) and/or posttranslational mechanisms. Recent evidence suggests that p27KlP1 protein may be a substrate for cyclin E/Cdk2 kinase activity and that the resulting phosphorylation may increase degradation of p27Kipl protein. Thus, increased levels of p27K,pl protein detected in differentiating trophoblasts may, in part, be a consequence of decreased cyclin E/Cdk2 catalytic activity. buy diabetes drugs

Expression of Gt Cyclins: DISCUSSION(2)

Many studies attribute decreased Cdk kinase activity in quiescent or differentiating cells to an increase in the expression of Cdk-inhibitory proteins (reviewed in ) including p27Kipl. Expression of p27KiP* gene and protein was apparent in freshly isolated trophoblasts, yet significantly decreased within the first 24 h after culture (Figs. 6b and 2c, respectively). Subsequently, expression of p27Kipi protein increased to levels 4-fold above those in freshly isolated trophoblast. birth control pills

Expression of Gt Cyclins: DISCUSSION(1)

DISCUSSION(1)

Expression of G[ cell cycle regulatory proteins, specifically cyclin E and D-type cyclins, is dictated by the stage of the cell cycle, cell or tissue type, and developmental stage of the tissue. Cyclin E expression and activation of its catalytic partner Cdk2 are required for progression of proliferating cells through Gb and cyclin E is rate-limiting for G,/S transition in both Rb-negative and Rb-positive cells. In our trophoblast culture sys-tern, levels of cyclin E protein steadily decreased from the time of isolation to 96 h in culture. Although cyclin E protein levels are substantial in comparison to those in MCF-7 breast cancer cells, a 3-fold or greater decrease in cyclin E protein expression was evident after 96 h of culture (Fig. 2a). ventolin inhaler

Expression of Gt Cyclins: RESULTS(7)

Messenger RNA for p27Kipl was present in freshly isolated cytotrophoblasts and declined after 24 h of culture (Fig. 6b). However, when normalized to actin mRNA, levels of the 2.5-kb transcript for p27KiP’ were up-regulated at 48, 72, and 96 h in culture to 2-, 2.5-, and 3-fold (respectively) that of freshly isolated trophoblasts (Fig. 6b). Levels of p27Kipl mRNA at 24 h in culture were 6- and 7.6-fold less than that in cultured trophoblasts at 72 and 96 h after culture, respectively.

Expression of Gt Cyclins: RESULTS(6)

RESULTS(6)

Associated with decreased Cdk2 kinase activity is a decrease in the slower-migrating, hyperphosphorylated form of the retinoblastoma tumor suppressor gene product, pRb (Fig. 4c). Western blot analyses of lysates from 24-h cultures indicate the presence of both hyperphosphorylated and hypophosphorylated pRb, similar to that of freshly isolated cytotrophoblasts (Fig. 4c). However, by 48 h and thereafter, the degree of phosphorylation of pRb was significantly reduced, perhaps suggesting a role for pRb in maintaining the differentiated state of the trophoblast.

Expression of Gt Cyclins: RESULTS(5)

Cdk2-Associated Kinase Activity and Cyclin E-Dependent Kinase Activity Related to Differentiation

Levels of the Cdk inhibitor p27Kipl increased and levels of cyclin E protein in complexes with Cdk2 were reduced in cultured trophoblasts, suggesting that Cdk2 function may be altered in differentiating trophoblast. Therefore, functional Cdk2-associated kinase activity was examined in freshly isolated cytotrophoblast and trophoblast lysates harvested at 24, 48, 72, and 96 h. Cell extracts were immu-noprecipitated with antibody to Cdk2 as described in Materials and Methods and assayed for kinase activity using histone HI as a substrate. Cdk2-associated kinase activity was greatest in freshly isolated cytotrophoblast and subsequently decreased (Fig. 4a). Cdk2-associated kinase activity was reduced between 24 and 48 h by an average of 55%. By 96 h in culture, trophoblast kinase activity was less than 20% of that found in freshly isolated trophoblasts. buy ampicillin

Expression of Gt Cyclins: RESULTS(4)

RESULTS(4)

D-type cyclins (Dl, D2, and D3) have been implicated in the regulation of G] progression, cell cycle withdrawal, and differentiation in numerous cell types. Therefore, in addition to cyclin E expression, we examined the expression of cyclins Dl, D2, and D3 in freshly isolated and cultured trophoblasts and proliferating ED27 and JEG-3 cells, as well as in MCF-7 breast cancer cells. As shown in Figure 2a, cyclin E expression was greatest in freshly isolated trophoblasts and trophoblasts cultured for 24 h (Fig. 3a). asthma inhalers

Expression of Gt Cyclins: RESULTS(3)

As positive controls for cyclin E expression, proliferating JEG-3 choriocarcinoma cells and ED27 cells were lysed, and equal amounts of protein were fractionated on reducing SDS gels along with freshly isolated term trophoblasts and trophoblasts cultured for 12 h (Fig. 2d). Western blot analyses demonstrated expression of large amounts of cyclin E in both JEG-3 and ED27 cells, similar to that of freshly isolated cytotrophoblast and short term-cultured trophoblasts (Fig. 2d). buy prednisone

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