Cell cycle regulation of human pancreatic cancer by tamoxifen

BACKGROUND: Clinical trials have suggested a survival advantage for selected patients with metastatic pancreatic cancer treated with tamoxifen. We sought to identify the molecular mechanism by which tamoxifen inhibits human pancreatic cancer cell (HPCC) growth. METHODS: HPCCs were grown in tamoxifen and growth inhibition was determined by 3H-thymidine uptake and by the MTT assay; changes in cell viability were determined by cell counts. Cell cycle alterations were evaluated by FACS, and the induction of apoptosis was evaluated using the TUNEL assay. Total cellular RNA was isolated after tamoxifen treatment, and Northern blot analysis was performed for p21waf1. RESULTS: Tamoxifen inhibited HPCC growth as measured by inhibition of 3H-thymidine incorporation and by the MTT assay. However, there was no decrease in the total number of viable cells after 6 days of treatment with 10 microM of tamoxifen and no evident apoptosis, confirming the absence of a cytotoxic effect. Cell cycle analysis revealed cellular arrest in the G0/G1 phase, which correlated with p21waf1 mRNA upregulation in response to tamoxifen treatment. CONCLUSIONS: Tamoxifen inhibits HPCC growth by inducing G0/G1 arrest with an associated increase in p21waf1 mRNA expression. Tamoxifen is an effective inhibitor of HPCC growth in vitro and warrants further in vivo study.     

Cell cycle regulation in irradiated and nonirradiated cells

Exposure of cells to ionizing radiation induces damage in the DNA. The adverse consequences of such exposures depend on the amount of the DNA damage induced as determined by the absorbed dose, as well as by its form as determined by the linear energy transfer. In addition to physical determinants, biological factors critically affect radiation response. Cells have the ability to sense DNA damage, and to activate repair pathways that efficiently remove such damage and restore the integrity of the DNA. Highly sophisticated mechanisms further enable cells to actively stall growth and division after sensing DNA damage. Such inhibitory processes, termed checkpoints, may optimize repair and minimize the adverse consequences of DNA damage. We review the fundamental principles underlying checkpoints as they have emerged from active research in the last few years and discuss briefly their relevance to the practice of medical and radiation oncology.     

G2 cell cycle arrest and apoptosis are induced in Burkitt''s lymphoma cells by the anticancer agent oracin

The gamma isoform of protein kinase C (gamma-PKC) activity is elevated and learning is superior in the inbred C57BL/6 mouse when compared to the DBA/2 mouse strain. Given the proposed link between PKC and long-term potentiation (LTP) on the one hand and PKC and learning on the other, it was predicted that LTP persistence would be greater in C57BL/6 mouse. When suprathreshold levels of tetanic stimulation were used, similar persistent LTP was observed in both C57BL/6 and DBA/2 strains. However, when tetanus was at threshold, the response in DBA/2 mice decayed to baseline in 30 min, similar to short-term potentiation (STP). Using this same paradigm with C57BL/6 mice, LTP persisted for 4 h, the longest time tested. The time course of the results parallels those observed in rat when phorbol ester, a potent PKC activator, converts STP to LTP. The present findings thus confirm the predicted difference between the two mouse strains. Moreover, the present findings are consistent with a role for gamma-PKC in LTP. Since such results call attention to the need for gamma-PKC interventive procedures, the relative utility of current PKC inhibitors, null mutants and antisense methods are discussed.     

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.     

Cell cycle gene regulation in reversibly differentiated new human hepatoma cell lines

We performed a comparative investigation of the immunomorphological characteristics of lymphatic and blood microvascular endothelial cells in normal human skin, cutaneous lymphangiomas, and hemangiomas, employing a pre-embedding immunogold electron microscopic technique. We stained for cell membrane proteins that are commonly used for light microscopic characterization of blood endothelial cells. With blood microvascular endothelial cells, we observed uniform labeling of the luminal cell membranes with monoclonal antibodies (MAbs) JC70 (CD31), EN-4 (CD31), BMA120, PAL-E, and QBEND-10 (CD34), and strong staining of the vascular basal lamina for Type IV collagen under normal and pathological conditions. In contrast, lymphatic microvascular endothelial cells in normal human skin and in lymphangiomas displayed, in addition to a luminal labeling, pronounced expression of CD31 and CD34 along the abluminal cell membranes. Moreover, CD31 was preferentially detected within intercellular junctions. The expression of CD34 was mostly confined to abluminal endothelial microprocesses and was upregulated in lymphangiomas and hemangiomas. Type IV collagen partially formed the luminal lining of initial lymphatics and occasionally formed bridges over interendothelial gaps. Our findings suggest a function of transmigration protein CD31 in recruitment of dendritic cells into the lymphatic vasculature. CD34 labeling may indicate early endothelial cell sprouting. The distribution of Type IV collagen also supports its role as a signal for migration and tube formation for lymphatic endothelial cells.     

Cell cycle regulation of the Saccharomyces cerevisiae polo-like kinase cdc5p

Progression through and completion of mitosis require the actions of the evolutionarily conserved Polo kinase. We have determined that the levels of Cdc5p, a Saccharomyces cerevisiae member of the Polo family of mitotic kinases, are cell cycle regulated. Cdc5p accumulates in the nuclei of G2/M-phase cells, and its levels decline dramatically as cells progress through anaphase and begin telophase. We report that Cdc5p levels are sensitive to mutations in key components of the anaphase-promoting complex (APC). We have determined that Cdc5p-associated kinase activity is restricted to G2/M and that this activity is posttranslationally regulated. These results further link the actions of the APC to the completion of mitosis and suggest possible roles for Cdc5p during progression through and completion of mitosis.     

Cell cycle regulation of the double stranded RNA activated protein kinase, PKR

The interferon (IFN)-induced, double stranded RNA (dsRNA)-activated serine/threonine kinase, PKR, is a potent negative regulator of cell growth when overexpressed in yeast or mammalian cells. To determine whether endogenous PKR plays a role in cell growth control, we have investigated the regulation of PKR levels and activity during the cell cycle in human glioblastoma T98G cells. The steady-state level of PKR mRNA in T98G cells was highest in growth arrested cells, dropped sharply within 3 h of serum stimulation then gradually increased as cells progressed through G1, reaching a plateau in early S phase. PKR protein level increased following serum stimulation reaching a peak at the G2+M boundary and declining thereafter. In contrast, PKR kinase activity exhibited two peaks, in early G1 and at the G1/S boundary, declining sharply in early S phase. Thus, the activity profile did not follow the protein profile indicating a tight regulation of PKR at the level of activity. In T98G cells expressing the catalytically inactive PKRK296R the dsRNA-induced activation of NF-kappaB and IRF-1 was suppressed and the mutant cells exhibited resistance to stress induced apoptosis. Cell cycle distribution analysis showed that the mutant expressing cells exhibited longer G1 phase and fewer cells engaged in S phase. Furthermore, early passage mouse embryo fibroblasts derived from PKR knockout mice grew more slowly compared with the control cells. Taken together these results suggest that PKR may play a role in cell cycle progression.     

Cell regulation

We describe the interaction of methyl isocyanate with reactive sulphydryl groups of rat erythrocyte membrane proteins. Intraperitoneal administration, as well as in vitro incubation of methyl isocyanate, caused a significant reduction in the free sulphydryl content of erythrocyte membrane proteins. [1-14C]Methyl isocyanate was specifically bound to band III of the erythrocyte membrane and caused the breakdown of band III proteins.     

Application of anticancer agent embolism in Miles'' operation

A double-blind prospective study was done with 15 patients with anterior shoulder instability to determine the diagnostic efficacy of magnetic resonance (MR) imaging versus arthroscopy in the evaluation of chondral or osteochondral lesions of the humeral head. MR produced 6 true positives, 5 true negatives and 4 false negatives, and its accuracy and sensitivity were 60% and 87%, respectively, whereas arthroscopy gave 8 true positives, 5 true negatives and 2 false negatives, with a sensitivity of 80% and an accuracy of 87%. All lesions diagnosed with either method were regarded as positive by definition, with the result that the specificity was always 100%. The differences in diagnosis sprang from the false negatives. The 40% discrepancy between the two methods was probably due to our distinction in MR between intra- and extra-articular osteochondral lesions. In the first group (the 4 MR false negatives), there were three instances of first-degree intra-articular lesion and one diagnostic error (third-degree lesion). In the second (the 2 arthroscopy false negatives), the lesions were of the extra-articular type. It is thus advisable to employ both of these methods to ensure the correct diagnosis of a Hill-Sachs lesion, and hence the correct choice of treatment.     

Endocrine regulation of the menstrual cycle

Physiological data concerning the serum concentrations and the pituitary content of varian steroids and gonadotropins, respectively, are reviewed. On the basis of these physiological data results from experimental studies are critically evaluated as far as their significance for the understanding of the physiology of the cycle is concerned. Data from the literatur and results from own studies are incorporated in a concept of the endocrine regulation of the human menstrual cycle.     

Cell cycle characteristics of thermophilic archaea

We have performed a cell cycle analysis of organisms from the Archaea domain. Exponentially growing cells of the thermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius were analyzed by flow cytometry, and several unusual cell cycle characteristics were found. The cells initiated chromosome replication shortly after cell division such that the proportion of cells with a single chromosome equivalent was low in the population. The postreplication period was found to be long; i.e., there was a considerable time interval from termination of chromosome replication until cell division. A further unusual feature was that cells in stationary phase contained two genome equivalents, showing that they entered the resting stage during the postreplication period. Also, a reduction in cellular light scatter was observed during entry into stationary phase, which appeared to reflect changes not only in cell size but also in morphology and/or composition. Finally, the in vivo organization of the chromosome DNA appeared to be different from that of eubacteria, as revealed by variation in the relative binding efficiency of different DNA stains.     

Hydra hymanae: regulation of the life cycle by time and temperature

Hydra hymanae, a hermaphroditic freshwater coelenterate, reproduces asexually at 24 degrees C and sexually at 15 degrees C. The appearance of gonads begins 12 days after transfer from 24 degrees to 15 degrees C and is complete 35 days after the temperature transition. Testes appear before eggs. Fifty percent of the mature embryos maintained at 15 degrees C hatch by day 61, but they have a low level of survival. Fifty percent of the mature embryos pretreated for from 5 to 25 days at 4 degrees C hatch by about day 45, and these have a high level of survival. Embryos maintained at 4 degrees C for longer periods (55 to 85 days) accumulate in a prehatching state and hatch with a high degree of synchrony approximately 7.5 days after return to 15 degrees C. Populations derived from newly hatched polyps are refractory to sex induction for approximately 120 days. The system is well adapted to ensure a regular alternation of reproductive modes in the natural environment.     

Taxol: the chemistry and structure-activity relationships of a novel anticancer agent

Taxol is an exciting new anticancer drug, showing clinical activity against ovarian and breast cancer. Its development as a clinically useful drug has involved major efforts to overcome the supply problem; this has now been done, and the focus of interest has moved to the development of improved analogs of the drug. Recent notable achievements include the first total synthesis of taxol, and the first indications of its binding site on tubulin.     

Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation

DNA polymerase alpha-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase alpha-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase alpha-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase alpha-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase alpha-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.