Impedance of a goat eye lens

Cyclins are essential proteins in cell cycle control, and their deranged expression has been reported to be associated with malignant transformation. Involvement of cyclins in the development of endometrioid carcinomas of the endometrium was studied immunohistochemically using antibodies against both cyclin A and tumor suppressor gene product p53, and their expression was compared with that of Ki-67 antigen. Sixty-two cases of endometrial endometrioid carcinoma and 20 cases of normal endometrium (10 proliferative and 10 secretory phase) were examined. Of the 62 endometrioid carcinomas, atrophic endometrium and hyperplasia were found adjacent to the cancers in 30 and 19 cases respectively. Cyclin A was expressed in < 1% of the glandular cells of normal endometrium in the proliferative phase and in hyperplasia, but was negligible in normal secretory phase and atrophic endometrium. p53 was almost always negative in normal endometrium and hyperplasia. Of the 62 endometrioid carcinomas, 12 tumors (19.4%) overexpressed cyclin A and 21 tumors (33.8%) overexpressed p53 (positive cells > 1%). Cyclin A and p53 were more frequently expressed in poorly differentiated tumors than in well differentiated tumors (cyclin A, p = 0.002; p53, p = 0.016). In addition, cyclin A-positive cells were topographically related to those cells positive for p53 as well as Ki-67. In conclusion, the abnormal expression of cyclin A and p53 is associated with high-grade endometrial endometrioid carcinomas.     

Exchange of Ser-4 for Val, Leu or Asn in the sequon Asn-Ala-Ser does not prevent N-glycosylation of the cell surface glycoprotein from Halobacterium halobium

The aim of the present study was to gain a better understanding of the localization of apoptotic cells within the human endometrium during the menstrual cycle and to elucidate the relationships among the following for the human endometrium: apoptosis, p21 expression, and cell proliferation. Apoptosis and p21 expression were identified mainly in the glandular cells of the basal layer in the late secretory phase. In contrast, cells positive for Ki-67 were observed predominantly in the functional layer (in the proliferative phase in glandular cells and in the secretory phase in stromal cells). A very strong positive correlation (r = 0.81; P < 0.001) was demonstrated between the number of apoptotic cells and the number of p21-positive cells present among the glandular cells but, topographically, individual apoptotic cells were not coincident with p21-positive cells in serial sections. The results of this study suggest that the proliferation of the glandular cells of the basal layer is regulated by both apoptosis and p21 expression, particularly in the late secretory phase. Such regulation may be necessary to maintain a healthy population of glandular cells in the basal layer of the endometrium.     

Reduced levels of the cell-cycle inhibitor p27Kip1 in epithelial dysplasia and carcinoma of the oral cavity

Recent studies have shown that the cyclin-dependent kinase (cdk) inhibitors play important roles in cell cycle progression in normal cells. Alterations in the cdk inhibitors also appear to be important in cancer development in a number of human tumors. p27Kip1 is a member of the CIP/KIP family of cdk inhibitors that negatively regulates cyclin-cdk complexes. Reduced levels of p27Kip1 protein have been identified in a number of human cancers, and in some cases reduced p27Kip1 is associated with an increase in proliferative fraction. In the present study, we examined p27Kip1 protein by immunohistochemistry in 10 normal and 36 dysplastic epithelia and in 8 squamous cell carcinomas from one anatomical site within the oral cavity, the floor of the mouth. Proliferative activity was assessed in serial sections by determining the expression of the cell cycle proteins Ki-67 and cyclin A. p27kip1 protein was significantly reduced in oral dysplasias and carcinomas compared with that in normal epithelial controls. In addition, there was a significant reduction in p27Kip1 protein between low- and high-grade dysplasias, suggesting that changes in p27Kip1 expression may be an early event in oral carcinogenesis. There was increasing expression of Ki-67 and cyclin A proteins with increasingly severe grades of dysplasia compared with normal controls. Although there was a strong correlation between Ki-67 and cyclin A scores (r2= 0.61) for all categories of disease, there was a weak negative correlation between Ki-67 and p27Kip1 levels (r2 = 0.29) and between cyclin A and p27Kip1 levels (r2 = 0.25). In conclusion, this study has found that a reduction in the proportion of cells expressing p27Kip1 protein is frequently associated with oral dysplasia and carcinoma from the floor of the mouth. Furthermore, reductions in p27Kip1 levels are associated with increased cell proliferation, although other changes likely contribute to altered cell kinetics during carcinogenesis at this site.     

Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis

The human endometrium undergoes a complex process of vascular and glandular proliferation, differentiation, and regeneration with each menstrual cycle in preparation for implantation. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic protein that appears to play an important role in both physiological and pathological neovascularization. To investigate whether VEGF may regulate human endometrial angiogenesis, we examined VEGF messenger ribonucleic acid (mRNA) and protein throughout the menstrual cycle and studied the regulation of VEGF by reproductive steroids in isolated human endometrial cells. By ribonuclease protection analysis, VEGF mRNA increased relative to early proliferative phase expression by 1.6-,2.0-, and 3.6-fold in midproliferative, late proliferative, and secretory endometrium, respectively. In histological sections, VEGF mRNA and protein were localized focally in glandular epithelial cells and more diffusely in surrounding stroma, with greatest VEGF expression in secretory endometrium. Consistent with these in vivo results, the treatment of isolated human endometrial cells with estradiol (E2), medroxyprogesterone acetate (MPA), or E2 plus MPA significantly increased VEGF mRNA expression over the control value by 3.1-, 2.8-, and 4.7-fold, respectively. The VEGF response to E2 was rapid, with steady state levels of VEGF mRNA reaching 85% maximum 1 h after the addition of steroid. E2 also caused a 46% increase in secreted VEGF protein, and the combination of E2 and MPA caused an 18% increase. VEGF expression in endometriosis, an angiogenesis-dependent, estrogen-sensitive disease was similar to that seen in eutopic endometrium. Peritoneal fluid concentrations of VEGF were significantly higher in women with moderate to severe endometriosis than in women with minimal to mild endometriosis or no disease. VEGF, therefore, may be important in both physiological and pathological angiogenesis of human endometrium, as it is an estrogen-responsive angiogenic factor that varies throughout the menstrual cycle and is elevated in women with endometriosis.     

Cell cycle regulation and differentiation in the human podocyte lineage

Mature podocytes are regarded as growth-arrested cells with characteristic phenotypic features that underlie their function. To determine the relationship between cell cycle regulation and differentiation, the spatiotemporal expression of cyclin A, cyclin B1, cyclin D1, the cyclin-dependent kinase inhibitors (CKIs) p27 and p57, and markers of differentiating podocytes in developing human kidneys was investigated by immunohistochemistry. In S-shaped body stage, Ki-67, a cell proliferation marker that labels the G1/S/G2/M phase, was expressed in the majority (more than 80%) of presumptive podocytes, along with cyclin A (approximately 20% of the Ki-67-positive cells) and cyclin B1 (less than 5% of Ki-67-positive cells) expression. Among these cells), cyclin D1 and CKIs were markedly down-regulated. At the capillary-loop stage, by contrast, CKIs and cyclin D1 were intensely positive in podocytes, whereas no Ki-67, cyclin B1, or cyclin A expression was seen. Moreover, double-immunolabeling and serial-section analysis provided evidence that CKIs and markers specific for differentiating podocytes, namely PHM-5 (podocalyxin-like protein in humans), synaptopodin (a foot process-related protein), and C3b receptor, were co-expressed at the capillary-loop stage. Podocytes were the only cells within the glomeruli that expressed CKIs at immunohistochemically detectable levels. Furthermore, bcl-2 (an apoptosis inhibitory protein) showed a reciprocal expression pattern to that of CKI. These results suggest that 1) the cell cycle of podocytes is regulated by cyclin and CKIs, 2) CKIs may act to arrest the cell cycle in podocytes at the capillary-loop stage, and 3) the specific cell cycle system in podocytes may be closely correlated with their terminal differentiation in humans.     

Cytoplasmic displacement of cyclin E-cdk2 inhibitors p21Cip1 and p27Kip1 in anchorage-independent cells

Loss of attachment to an extracellular matrix substrate arrests the growth of untransformed cells in the G1 phase. This anchorage-dependent cell cycle arrest is linked to increased expression of the p21Cip1 (p21) and p27Kip1 (p27) cyclin-dependent kinase inhibitors. The result is a loss of cdk2-associated kinase activity, especially that of cyclin E-cdk2. The levels of p21 and p27 are also upregulated in unattached transformed cells, but cyclin E-cdk2 activity remains high, and the cells are able to grow in an anchorage-independent manner. Increased expression of cyclin E and cdk2 appears to be partially responsible for the maintenance of cyclin E-cdk2 activity in transformed cells. To explore further the regulation of cyclin E-cdk2 in transformed cells, we have analysed the subcellular distribution of cyclin-cdk complexes and their inhibitors in normal human fibroblasts, their transformed counterparts, and in various human tumor cell lines. In substrate-attached normal fibroblasts, cyclin E and cdk2 were exclusively in the nuclear fraction, associated with one another. When normal fibroblasts were detached and held in suspension, cyclin E-cdk2 complexes remained nuclear, but were now found associated with the p21 and p27 cdk inhibitors and lacked histone H1 phosphorylating activity. In contrast, the transformed fibroblasts and tumor cells, which are anchorage-independent, had more than half of their cyclin E, cdk2, p21 and p27 in the cytoplasmic fraction, both in attached and suspended cultures. The cytoplasmic p21 and p27 were bound to cyclin E-cdk2, as well as to complexes containing cyclin A and cyclin D. The nuclear cyclin E-cdk2 complexes from the transformed cells grown in suspension contained only low levels of p21 and p27 and had histone H1 kinase activity. Thus, at least three mechanisms contribute to keeping cyclin E-cdk2 complexes active in suspended anchorage-independent cells: cyclin E and cdk2 are upregulated, as reported previously, cdk inhibitors are sequestered away from the nucleus by cytoplasmic cyclin-cdk complexes, and the binding of the inhibitors to nuclear cyclin E-cdk2 complexes is impaired.     

Compartmentalization and cyclic variation of immunoreactivity of renin and angiotensin converting enzyme in human endometrium throughout the menstrual cycle

Renin and angiotensin converting enzymes (ACE) are responsible for the generation of angiotensin II which regulates blood pressure and fluid/electrolyte homeostasis. The cellular localization and cyclic distribution of renin and ACE in human endometrium are demonstrated in this study. Immunohistochemical studies revealed that both renin and ACE were consistently localized in the endometrial glandular epithelia throughout the menstrual cycle; however, the immunostainings respectively for ACE and renin were weak and moderate in stromal cells of proliferative endometrium and negligible in secretory endometrium. No renin immunostaining was detected around endometrial blood vessels. Although endothelial cells consistently stained for ACE, no renin immunoreactivity was detected in these cells during the menstrual cycle. Western blot analysis using ACE antibody directed against human kidney identified a single protein band with a relative molecular mass of approximately 153 kDa. The intensity of this band showed cyclic variation during the menstrual cycle with the highest ACE expression during the late secretory phase and at menses suggesting that ACE plays a role in the initiation of menstruation. The differences in the cellular distribution patterns of these two enzymes further supports our previous proposition that angiotensin II has different functions at the different stages of the menstrual cycle.     

Identification and characterization of cytosolic sulfotransferases in normal human endometrium

Understanding the factors which alter estrogen metabolism and activity in endometrial tissue is important because unopposed estrogen stimulation is an important risk factor in the development of endometrial carcinoma. The cyclic progression of the endometrium through proliferative and secretory phases is normally under the control of the ovarian hormones beta-estradiol (E2) and progesterone. One mechanism by which progesterone inhibits the activity of E2 in secretory endometrium is by elevating the degree of E2 sulfation, thereby reducing its ability to bind to the estrogen receptor and elicit a cellular response. Our laboratories have investigated the cytosolic sulfotransferases (STs) found in biopsies of both proliferative and secretory endometrium obtained from five normal pre-menopausal women who were not taking any drugs or steroids. Two of the human cytosolic STs were detected in human endometrial tissues. The phenol-sulfating form of phenol ST (P-PST) was found at varying levels in cytosol from both proliferative and secretory endometrium in all of the women studied but with no consistent correlation to the phase of the menstrual cycle. In contrast, estrogen ST (EST) was not detected in the proliferative endometrial cytosol of any of the women studied but was consistently found in all of the secretory endometrial cytosols. The presence and levels of these STs was confirmed by ST activity studies, immunoblot analysis and Northern blot analysis. These results indicate that the expression of EST in human endometrial tissues varies with the phase of the menstrual cycle and is most likely regulated by progesterone secreted from the ovaries.     

Sex-role reversal and the absence of extra-pair fertilization in Wilson''s phalaropes

Human endometrial leukocytes undergo regular cyclical changes during the menstrual cycle, with a striking increase in the phenotypically unusual population of CD56+ CD16- endometrial granulated lymphocytes (eGLs) in the late secretory phase and early pregnancy. The factors that regulate this increase in eGL numbers are unclear; their unusual morphology, however, has led to the suggestion that they undergo apoptosis at the end of the menstrual cycle. Apoptosis, bcl-2 expression, and proliferative activity were examined in the stroma of normal cycling, progesterone-treated, and early-pregnancy endometrium. The expression of bcl-2 and the Ki67 proliferation marker by highly purified (> 98% CD56+) eGLs from endometrium during the menstrual cycle and from first-trimester decidua was also studied. Apoptotic cells were rarely observed in the endometrial stroma of any of the samples examined. Stromal bcl-2 expression, however, increased from the proliferative to the premenstrual phase, and double immunohistochemical labeling demonstrated large numbers of bcl-2+ CD56+ eGLs. In contrast, Ki67 expression was high in the endometrial stroma during the proliferative phase, fell during the secretory phase, and rose again premenstrually, because of expression by eGLs. Isolated CD56+ eGLs also showed high bcl-2 and Ki67 expression at the end of the menstrual cycle. Unlike premenstrual endometrium, progesterone-treated endometrium and first-trimester decidua contained few proliferating cells, expressed high levels of bcl-2, and showed no evidence of apoptosis. Thus, eGLs do not undergo apoptosis in premenstrual endometrium, and their regulatory mechanisms remain to be clarified.     

Expression of telomerase activity in human endometrium is localized to epithelial glandular cells and regulated in a menstrual phase-dependent manner correlated with cell proliferation

Telomerase activity is observed in most malignant tumors and germ cells, whereas normal somatic cells usually do not express it. Human endometrium is composed of glandular and stromal components and exhibits dramatic changes in proliferative activity during the menstrual cycle, which is exquisitely regulated by estrogen function. We previously reported that normal human endometrium expresses telomerase activity. However, it remains unclear which of the above components are the major sources of telomerase activity and how levels of telomerase activity are regulated over the menstrual cycle. Quantitative analysis of telomerase activity revealed that it changes dramatically over the course of the menstrual cycle and is strictly regulated in a menstrual-phase-dependent manner. Maximal activity equivalent to that in endometrial cancer was present in late proliferative phase, and minimal activity in late secretory phase. Postmenopausal endometrium and endometrium treated with anti-estrogen drugs exhibited decreased telomerase activity. Testing isolated epithelial glandular cells and stromal cells, we found that telomerase activity was localized to epithelial glandular cells. In situ RNA hybridization analysis also revealed epithelial-specific expression of human telomerase RNA. In vitro analysis of cultured epithelial cells demonstrated that telomerase activity is correlated with epithelial proliferation but not affected by estrogen treatment. These findings suggest that expression of telomerase activity is specific to epithelial cells and linked to cell proliferative status. The involvement of estrogen in telomerase regulation remains to be elucidated.     

The CDK inhibitor, p27Kip1, is required for IL-4 regulation of astrocyte proliferation

IL-4 is a pleiotrophic cytokine that has been shown to affect cells of the central nervous system. We have demonstrated that IL-4 inhibits DNA synthesis and proliferation in human astroglia expressing IL-4 receptors. In this study, we sought to identify mechanisms that could account for the antimitogenic effects of IL-4. Epidermal growth factor (EGF)-stimulated human astroglia were arrested in G1 phase by IL-4, even though IL-4 stimulated levels of the G1 cyclins, D1 and E. Histone H1 kinase activity of cdk2 immunoprecipitates, however, was sharply reduced by IL-4; impairment of kinase activity was also evident in cyclin E immunoprecipitates, which contained evidence of hypophosphorylated (inactive) cdk2 product. Reduced cyclin E-associated cdk2 activity was not due to impaired cyclin-dependent kinase-activating kinase (CAK) activity, which was unaffected by IL-4. Inactive cyclin E/cdk2 complexes from IL-4 + EGF-treated cells contained, however, strikingly elevated p27Kip1 cdk inhibitor. Elevated p27 was also detectable in whole cell lysates after 24 and 48 h of IL-4 treatment; by 72 h, p27 was no longer elevated. Pretreatment with antisense but not mismatch p27 oligonucleotides attenuated the inhibitory effects of IL-4 on DNA synthesis and histone kinase activity of cyclin E/cdk2 complexes. Antisense p27 also abrogated IL-4-mediated elevation of p27 in whole cell lysates and cyclin E/cdk2 complexes. These findings demonstrate that IL-4 regulates the cell cycle machinery of astroglial cells via a p27Kip1 braking mechanism.     

Response of the primate secretory endometrium to subchronic hypercortisolemia

OBJECTIVE: To assess the impact of subchronic and moderate hypercortisolism on the secretory endometrium of the cynomolgus monkey. METHODS: Osmotic pumps containing hydrocortisone phosphate (HP) were implanted subcutaneously in each monkey on the first day of the menstrual cycle; each monkey also received pumps containing saline in another cycle. Blood was obtained three times per week and urine was collected daily for hormone analyses. Endometriectomy was performed 13 +/- 1 days after the serum estradiol (E2) peak in each study cycle. RESULTS: Infusion of HP elevated serum cortisol levels by an average of 70%. Mean serum progesterone (P) levels were decreased by 50% during the secretory phase of HP-treatment cycles by comparison with self-control cycles (P < .01); as a result, the mean endometrial glycogen concentration was reduced by 30% (P < .05) and the activity of 17 beta-hydroxysteroid dehydrogenase was decreased by 70% (P < .05). Serum E2 levels were not consistently elevated by HP treatment, but cytosolic estrogen receptor levels of the endometrium were decreased by 50% (P < .01), indicating increased estrogenic stimulation. Histologic development of the secretory endometrium was retarded, but the length of the secretory phase was not affected by the treatment. CONCLUSION: A moderate elevation of serum cortisol levels over one menstrual cycle consistently produced a reduction in serum P and a hypoprogestogenic-hyperestrogenic response of the secretory endometrium in the cynomolgus monkey.     

Immunolocalization of inhibin and activin subunits in human endometrium across the menstrual cycle

Inhibin/activin alphaC/alphaN and betaA subunits were localized immunohistochemically in the human endometrium throughout the menstrual cycle using an affinity-purified sheep polyclonal antibody raised against the alphaC/alphaN subunit and an affinity-purified rabbit polyclonal antibody raised against the betaA subunit. The betaB subunit was below the level of detection in all human endometrial samples tested. Immunoreactive inhibin alphaC/alphaN subunit was localized in the luminal epithelium, glandular epithelium, stromal tissues and vascular endothelium with no significant variation across the normal menstrual cycle. Immunoreactive betaA subunit, common to inhibin A and activins AA and AB was localized in the luminal and glandular epithelium and in migratory cells while the endometrial stromal cells, decidua, vascular smooth muscle and endothelium were devoid of immunoreactivity. A significant variation of immunoreactive betaA subunit was observed in glandular and luminal epithelium across the normal menstrual cycle. In proliferative endometrium, only a very low level of betaA immunostaining was seen in luminal and glandular epithelium, while the luminal epithelial staining increased significantly in the early secretory phase and remained relatively constant over the rest of the menstrual cycle. A progressive increase in betaA immunoreactivity was observed also in the glandular epithelium during the secretory phase reaching a maximum in the late secretory phases, and decreasing at menstruation. Co-localization studies on serial sections suggested that the migratory cells expressing strong betaA immunoreactivity were macrophages and neutrophils but not eosinophils or mast cells. Thus, cells within the human endometrium are capable of expressing inhibin/activin molecules in vivo. The variation in the pattern of secretion of the betaA subunit across the menstrual cycle suggests that activin peptides may have a physiological role in endometrial function.     

Progesterone induces calcitonin gene expression in human endometrium within the putative window of implantation

The human endometrium acquires the ability to implant the developing embryo within a specific time window that is thought to open between days 19-24 of the secretory phase of the menstrual cycle. During this period the endometrium undergoes pronounced structural and functional changes induced by the ovarian steroids, estrogen and progesterone, that prepare it to be receptive to invasion by the embryo. The identification of reliable biochemical markers to assess this critical receptive phase in the context of the natural cycle remains one of the major challenges in the study of human reproduction. Our previous studies in a rat model system demonstrated that the expression of calcitonin, a peptide hormone involved in calcium homeostasis, is transiently induced by progesterone in the glandular epithelium at the onset of implantation. Attenuation of calcitonin synthesis in the uterus during the preimplantation phase by administration of calcitonin antisense oligodeoxynucleotides severely impairs implantation of rat embryos, suggesting that this peptide hormone plays a critical role in uterine receptivity. To investigate whether calcitonin is also expressed in the human endometrium during implantation, we monitored the spatio-temporal expression of calcitonin on various days of the menstrual cycle. Our studies employing RT-PCR showed that calcitonin messenger ribonucleic acid is expressed in human endometrium during the postovulatory midsecretory phase (days 17-25) of the menstrual cycle, with maximal expression occurring between days 19-21. Very little calcitonin expression was detected in the endometrium in either the preovulatory proliferative (days 5-14) or the late secretory (days 26-28) phase. In situ hybridization and immunocytochemical analyses localized the calcitonin expression predominantly in the glandular epithelial cells of the endometrium. Our studies further showed that calcitonin expression in the human endometrium is under progesterone regulation. Treatment of women with an antiprogestin, mifepristone (RU-486), drastically reduced calcitonin expression in the endometrium. Collectively, these findings reveal that progesterone-induced expression of calcitonin in the secretory endometrium temporally coincides with the putative window of implantation in the human.     

Regulated ectopic expression of cyclin D1 induces transcriptional activation of the cdk inhibitor p21 gene without altering cell cycle progression

Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and overexpressed in many cancers. To address the relationship between cyclin D1 and other cell cycle regulatory proteins, we established human glioma and rodent fibroblast cell lines in which cyclin D1 expression could be regulated ectopically with tetracycline. In both of these cell lines, we found that ectopic expression of cyclin D1 in asynchronously growing cells was accompanied by increased levels of the p53 tumor suppressor protein and the cyclin/cdk inhibitor p21. Despite the induction of these cell cycle inhibitory proteins, cyclin D1-associated cdk kinase remained activated and the cells grew essentially like that of the parent cells. Although growth parameters were unchanged in these cells, morphological changes were clearly identifiable and anchorage independent growth was observed in NIH3T3 cells. In a first step toward elaborating the mechanism for cyclin D1-mediated induction of p21 gene expression we show that co-expression of E2F-1 and DP-1 can specifically transactivate the p21 promoter. In support of these findings and a direct effect of E2F on induction of p21 gene expression a putative E2F binding site was identified within the p21 promoter. In summary, our results demonstrate that ectopic expression of cyclin D1 can induce gene expression of the cdk inhibitor p21 through an E2F mechanism the consequences of which are not to growth arrest cells but possibly to stabilize cyclin D1/cdk function.     

Placental protein 14 in endometrium during menstrual cycle and effect of early luteal phase mifepristone administration on its expression in implantation stage endometrium in the rhesus monkey

Placental protein 14 (PP14) is a glycoprotein which is secreted by secretory phase endometrium and decidua in women. Despite the suggestion that PP14 is involved in the process of endometrial maturation for blastocyst implantation, our understanding in this regard is poor. In the present study, the concentrations and distribution patterns of immunodetectable PP14 in the endometrium during proliferative and secretory phases of normal ovulatory menstrual cycles, as well as in implantation stage endometrium in naturally mated ovulatory cycles with or without early luteal phase mifepristone treatment, were investigated using the rhesus monkey as a primate model. Immunopositive PP14 was observed mainly in epithelial cells of glands and it was detected in one major immunopositive band at Mr 28 kDa in tissue homogenate and spent medium. The area of immunopositive precipitation of PP14 in glands was minimal in follicular phase endometrium, and was higher (P < 0.01) in early, mid- and late luteal phase endometrium compared with that in pre- and periovulatory phases of the cycle, but there was no change in its area profile in the glandular compartment throughout the luteal phase. Immunopositivity for PP14 in luminal contents of gland displayed an increasing profile from early to late secretory phases. Thus, the concentrations and the distribution of immunodetectable PP14 in luteal phase endometrium of the rhesus monkey showed marked similarity with those of human endometrium during the natural menstrual cycle. Although there was no marked change in the band characterstics for the protein in implantation stage endometrium following early luteal phase mifepristone treatment, it was markedly decreased (P < 0.01) in tissue homogenate and in vitro spent medium along with a lesser (P < 0.02) degree of immunoprecipitation in the glands in implantation stage samples of mifepristone treatment group compared with that in control group samples. Thus, the contragestional effect of early luteal phase mifepristone treatment appears to be associated with a decrease in the concentration of immunodetectable PP14 in implantation stage endometrial glands and its secretion in the rhesus monkey. It remains to be seen whether this decline is caused from direct antiprogesterone action on endometrial glands during progesterone dominance, or secondarily from associated retarded development of endometrium.     

CD40-mediated induction of p21 accumulation in resting and cycling B cells

The accumulation of G1 cell cycle-related proteins by resting or cycling B cells stimulated with B cell antigen receptor (BCR)- and T helper (Th) cell-derived signals is documented. Resting B cells constitutively express cyclin dependent kinase (cdk)4, cdk2 and the cyclin dependent kinase inhibitor (CKI), p27. The initiation of optimal proliferation with F(ab')2 anti-mu plus paraformaldehyde-fixed CD40 ligand-baculovirus-infected Sf9 cells (CD40L/Sf9 cells) increases accumulation of both cdk4 and cdk2 while decreasing p27 levels. B cells express cyclin D2 early during cycle progression, while cyclin D3 and E are not expressed until 18 h poststimulation and cyclin A by 24 h poststimulation. Cycling B cells express heightened levels of all these cyclins and cdks. Although neither BCR- nor CD40-mediated signals appreciably alter cycling B cell accumulation of cyclins D2, cdk4 and cdk2, the absence of BCR-derived signals results in a decreased accumulation of cyclins D3 and E. Finally, CD40-mediated signals induce resting B cells to accumulate the CKI, p21, while cycling B cells require both BCR- and CD40-mediated signals to maintain increased expression of p21. Thus, a Th cell-derived signal may impact upon both resting and cycling B cell cycle progression, at least in part, by regulating the accumulation of p21. The functional consequences of p21 accumulation as cells enter and move through the cell cycle are discussed.     

Changes in cyclins and cyclin-dependent kinases induced by DNA damaging agents in a human ovarian cancer cell line expressing mutated or wild-type P53

The expression of cyclins, cyclin-dependent kinases (cdk), and cdk inhibitors was evaluated in clones from a human ovarian cancer cell line transfected with a temperature-sensitive mutant of p53, after treatment with the anticancer agents doxorubicin (DX) and AMSA. The two drugs were selected on the basis of their activity in these clones, since AMSA is equally active in cells expressing mutated or wild-type (wt) p53, while DX was much less cytotoxic in cells expressing wt p53. In untreated cells, the expression of wt p53 induced an accumulation of cells in the G2 and perhaps also the G1 phase of the cell cycle. Concomitantly cyclin B1 and cdc2 increased. Cyclin E and particularly D1 levels were also raised by wt p53 expression. Treatment of mutated p53-expressing cells (SK23a cells kept at 37 degrees C) with DX or, more so, with AMSA, resulted in a strong accumulation of cyclin B1 and cdc2, in accordance with their ability to block cells in G2 phase of the cell cycle. Wt p53-expressing cells (SK23a cells kept at 32 degrees C) treated with the drugs showed an increase in p21 expression and consequently decreased kinase activity after immunoprecipitation with p21 antibodies. Cdc2-associated kinase activity was also reduced in these conditions. We could also observe a decrease in the percentage of cells in G1 and G2 phases and an accumulation of cells in S phase after both DX and AMSA. Cdk2, retinoblastoma, and p27 levels did not change significantly. Treatment with DX or AMSA caused similar effects, suggesting that p53-induced changes in cyclin, cdk, and cdk inhibitors after DNA damage are not responsible for the marked reduction in the cytotoxicity of DX we observed in wt p53-expressing cells.     

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 --> S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.