The HPV E7 oncoprotein inhibits tumor necrosis factor alpha-mediated apoptosis in normal human fibroblasts.

Tumor necrosis factor-alpha (TNF) is a cytokine that induces programmed cell death, apoptosis, in a number of cell types and is employed by cytotoxic T cells to eliminate virus infected cells. Consequently, many viruses have acquired mechanisms to undermine these host cell defense mechanisms and cause resistance to TNF-mediated apoptosis. Here we show that normal human diploid fibroblasts that express the human papillomavirus type 16 E7 oncoprotein have a decreased propensity to undergo apoptosis in response to TNF treatment. The ability of E7 to undermine TNF-mediated apoptosis correlates with cellular transformation. While E7 does not generally subvert signaling by tumor necrosis factor receptor 1, pro-caspase 8 activation is decreased in E7-expressing cells. E7 also provides some protection from apoptosis caused by stimulation of the TNF receptor 1-related cytokine receptor Fas, where induction of apoptosis occurs much slower in this cell type. Hence, E7-expressing normal human fibroblasts exhibit a specific defect that obstructs cytokine-mediated activation of pro-caspase 8 and apoptosis.

Production and characterization of an antiserum which recognizes the native receptor for thyrotropin-releasing hormone.

Despite attempts in several laboratories, it has been difficult to prepare antiserum to the thyrotropin-releasing hormone receptor (TRHR). We have prepared a polyclonal anti-rat TRHR antiserum by immunization of rabbits with a synthetic peptide corresponding to the C-terminus of the TRHR. The specificity of the antiserum was assessed by enzyme-linked immunosorbent assay. The affinity-purified antibody recognized a major broad band at 50-60 kDa and a minor broad band at 100-120 kDa in Western blot analysis of membrane proteins from TRHR-transfected, but not control, HEK293t cells. Binding to both bands was abolished by preincubation with the immunizing peptide but not control peptide. The approach was repeated with rat pituitary F4C1 cells, which lack endogenous TRHRs; membranes from F4C1 cells transfected with TRHR cDNA, but not control cells, showed specific binding by Western blot. Using laser confocal microscopy, the TRHR was visualized on the plasma membrane of transfected, but not control, F4C1 cells. Similar confocal findings were observed in TRHR-transfected HEK293t cells. Within 5 min after TRH addition, the TRHR signal translocated from the plasma membrane to the cytoplasm of F4C1 cells transfected with TRHR cDNA. Ten minutes after TRH addition, the TRHR signal formed aggregates in the cytoplasm. Thirty minutes after TRH treatment, both cytoplasmic and plasma membrane localizations were observed, suggesting recycling of some TRHRs back to the plasma membrane. These observations are consistent with our previous findings using an epitope-tagged TRHR. In conclusion, we have prepared an antiserum that recognizes the native TRHR by Western blot analysis and confocal microscopy.

Direct measurement of hormone-induced acidification in intact bone.

Previous findings have shown that osteoblasts respond to parathyroid hormone (PTH) with an increase in extracellular acidification rate (ECAR) in addition to the known effect of PTH to increase local acidification by osteoclasts. We, therefore, investigated use of the Cytosensor to measure the ECAR response of whole intact bone to PTH employing microphysiometry. The Cytosensor measures a generic metabolic increase of cells to various agents. Using neonatal mouse calvaria, we found that the area surrounding the sagittal suture was particularly responsive to PTH. In this bone, the increase in ECAR was slower to develop (6 minutes) and more persistent than in cultured human osteoblast-like SaOS-2 cells and was preceded by a brief decrease in ECAR. Salmon calcitonin also produced an increase in ECAR in this tissue but with a different pattern than that elicited by PTH. Because PTH stimulates osteoclastic bone resorption in mouse calvaria via a cyclic adenosine monophosphate (cAMP)-mediated mechanism, we showed that the adenylyl cyclase activator forskolin also stimulated ECAR in this tissue. When the protein kinase A (PKA) pathway was activated by maintaining a high intracellular concentration of cAMP using N6-2'-0-dibutyryladenosine-cAMP (db-cAMP), there was a reduction of PTH-induced acidification, while isobutylmethylxanthine pretreatment potentiated the PTH-induced acidification, consistent with a PKA-mediated pathway. Thapsigargin and the protein kinase C (PKC) activator phorbol myristate acetate had no effect on the PTH-induced increase in ECAR in calvaria, indicating that PKC does not play a major role in the ECAR response in intact bone. These results indicate the utility of using microphysiometry to study ECAR responses in intact tissue and should enable elucidation of the relative importance of extracellular acidification by osteoblasts and osteoclasts to the anabolic and catabolic activities of PTH, respectively.

Ca(2+) and extracellular acidification rate responses to parathyroid hormone fragments in rat ROS 17/2 and human SaOS-2 cells.

To examine the importance of the N- or C-termini of PTH(1-34) the effects of truncated fragments of PTH on human receptors in osteoblast-like SaOS-2 cells and rat receptors in rats ROS 17/2 cells were examined. Fura-2-loaded cells were used to monitor cytosolic free Ca(2+) concentration ([Ca2+]i), and the Cytosensor microphysiometer was used to monitor extracellular acidification rate (ECAR). C-terminally truncated fragments (1-31) and (1-28) of hPTH(1-34)NH(2) stimulated an increase in [Ca(2+)](i) and ECAR in both cell lines. hPTH(3-34)NH(2) and other N-terminally truncated fragments did not stimulate [Ca(2+)](i) or ECAR in either cell type. The signal transduction pathway of PTH-induced ECAR in ROS 17/2 cells was investigated to compare with previous results in SaOS-2 cells. Potentiation by IBMX, attenuation by 8Br-cAMP and lack of effect of the PKC inhibitor chelerythrine chloride support a cAMP/PKA-mediated signal transduction pathway in ROS 17/2, while the protein kinase C pathway was predominant in SaOS-2 cells. We conclude that the intact N-terminus of PTH is essential in PTH signaling mediated via either the cAMP/PKA or inositol lipid/Ca(2+)/PKC pathways in osteoblast-like cells.

A new action of parathyroid hormone. receptor-mediated stimulation of extracellular acidification in human osteoblast-like SaOS-2 cells.

The major physiological function of parathyroid hormone (PTH) is the maintenance of Ca2+/Pi homeostasis via the parathyroid hormone/parathyroid hormone-related protein receptor (PTHR) in kidney and bone. An important consequence of PTHR activation in bone is enhanced local acidification of the extracellular space. Agonist activation of some seven transmembrane-domain receptors increases the extracellular acidification rate (ECAR). We utilized microphysiometry to investigate PTH-stimulated, receptor-mediated increases in ECAR in human osteoblast-like SaOS-2 cells. PTH-(1-34) elicited a large, acute, dose-dependent increase in ECAR with an EC50 of about 2 nM. The PTH-induced increase in ECAR was specific to cells expressing the PTHR and was inhibited by PTHR antagonists. Rapid, partial, homologous desensitization of the PTH-induced increase in ECAR was observed. Incubation of SaOS-2 cells with 8-bromo-cyclic AMP neither mimicked nor abrogated the PTH effect, and PTH stimulated an acute increase in ECAR in cAMP-resistant SaOS-2 Ca#4A cells. Stimulation of ECAR by PTH was independent of transient increases in cytosolic free calcium. Both inhibition and down-regulation of PKC reduced the PTH-induced increase in ECAR. Inhibition of Na+/H+ exchange did not affect the PTH-induced ECAR response. We conclude that PTH caused a receptor-mediated, concentration-dependent, increase in ECAR, which was not dependent on the cAMP/PKA signaling pathway or the Na+/H+ exchanger but involved the action of PKC. Thus, acid production in bone, a physiologically important action of PTH, is not confined to osteoclasts as previously considered but is also mediated by osteoblasts.

The human papillomavirus-16 E6 oncoprotein decreases the vigilance of mitotic checkpoints.

The E6 and E7 proteins of the high risk human papillomaviruses (HPVs) are consistently expressed in HPV-positive cervical carcinomas. We investigated the ability of HPV-16 E6 and E7 to disrupt mitotic checkpoints in normal diploid human cells. Acute expression of HPV-16 E6, but not HPV-16 E7, decreased the fidelity of multiple checkpoints controlling entry into and exit from mitosis. After irradiation, nearly 50% of cells containing HPV-16 E6 readily entered mitosis as opposed to less than 10% of control cells. Consistent with this, asynchronous populations of cells expressing HPV-16 E6 had increased cdc2-associated histone H1 kinase activity relative to control populations. In addition, HPV-16 E6 increased sensitivity to chemically-induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function relative to control populations. HPV-16 E6 mutants with a reduced ability to target p53 for degradation were unable to abrogate mitotic checkpoints, suggesting a possible mechanism by which HPV-16 E6 disrupts mitotic checkpoints. Expression of a mutant p53 gene yielded an intermediate phenotype relative to HPV-16 E6, generating moderate increases in sensitivity to chemically-induced S-phase PCC and mitotic spindle disruption and a heightened propensity to enter mitosis after irradiation.

Premature expression of cyclin B sensitizes human HT1080 cells to caffeine-induced premature mitosis.

Eukaryotic cells do not normally initiate mitosis when DNA replication is blocked. This cell cycle checkpoint can be bypassed in some cells, however, by treatment with caffeine and certain other chemicals. Although S-phase arrested hamster cells undergo mitosis-specific events such as premature chromosome condensation (PCC) and nuclear envelope disassembly when exposed to caffeine, human cells show little response under the same conditions. To further investigate the molecular basis of this cell type specificity, a panel of hamster/human whole cell hybrids was created. The frequency of caffeine-induced PCC and the level of cyclin-B-associated H1 kinase activity in the various hybrids were directly correlated with the extent of cyclin B synthesis during S-phase arrest. To determine whether expression of cyclin B alone could sensitize human cells to caffeine, cyclin B1 was transiently overexpressed in S-phase arrested HT1080 cells. The transfected cell population displayed a 5-fold increase in the frequency of caffeine-induced PCC when compared with normal HT1080 cells, roughly equivalent to the frequency of cells expressing exogenous epitope-tagged cyclin B1. In addition, immunofluorescent microscopy showed that individual cells overexpressing cyclin B1 during S phase arrest underwent PCC when exposed to caffeine. These results provide direct evidence that premature expression of cyclin B1 can make cells more vulnerable to chemically-induced uncoupling of mitosis from the completion of DNA replication.

Tyrosine phosphorylations specific to mitosis in human and hamster cells.

Changes in protein tyrosine phosphorylation are known to be important for regulating cell cycle progression. With the aim of identifying new proteins involved in the regulation of mitosis, we used an antibody against phosphotyrosine to analyze proteins from synchronized human and hamster cells. At least seven proteins were found that displayed mitosis-specific tyrosine phosphorylation in HeLa cells (pp165, 205, 240, 250, 270, 290, and approximately 400) and one such protein in hamster BHK cells (pp155). In synchronized HeLa and BHK cells, all proteins except HeLa pp165, pp205, and pp250 were readily detectable only in mitosis. Tyrosine phosphorylation of pp165, pp205, and pp250 was apparent during arrest in S phase, suggesting that cell cycle perturbations can affect the phosphorylation state of some of these proteins. In a related finding in BHK cells, pp155 underwent tyrosine phosphorylation when cells were forced into premature mitosis by caffeine treatment. Only one protein (pp135 in HeLa cells) was found to be dephosphorylated on tyrosine during mitosis. The above findings may prove helpful for isolating new cell cycle proteins that are important for both the normal regulation of mitosis and the mitotic aberrations associated with cell cycle perturbations and chemical treatments.

Chemically induced premature mitosis: differential response in rodent and human cells and the relationship to cyclin B synthesis and p34cdc2/cyclin B complex formation.

Normal eukaryotic cells do not initiate mitosis until DNA replication has been completed. This requirement can be bypassed by exposing cells to certain chemicals. We report here that chemically induced premature mitosis is not readily achieved in all mammalian species. Although hamster cells underwent premature mitosis following treatment with caffeine, the protein phosphatase inhibitor okadaic acid, and the protein kinase inhibitors 2-aminopurine and 6-dimethyl-aminopurine, the mouse and human cells examined in this study displayed little or no response to any of these compounds. Differences in cell permeability or metabolism could not account for the species specificity of these drugs, because other biochemical and mitosis-promoting activities were apparent in human cells. Cell-type specificity can be explained, however, by the timing of cyclin B synthesis and p34cdc2/cyclin B complex formation during the cell cycle. Synthesis of cyclin B and formation of a p34cdc2/cyclin B complex, both of which are required for initiation of mitosis, were prevalent in hamster cells arrested in S phase but were absent or barely detectable in arrested human cells. In hamster cells, the hyperphosphorylated form of p34cdc2 was complexed with cyclin B and underwent tyrosine dephosphorylation during caffeine-induced premature mitosis. These findings indicate that the onset of mitosis is regulated somewhat differently among mammalian cell types and that these differences affect the vulnerability of cells to drug-induced mitotic aberrations and cytogenetic damage.

Premature mitosis induced in mammalian cells by the protein kinase inhibitors 2-aminopurine and 6-dimethylaminopurine.

The protein kinase inhibitors 2-aminopurine (2-AP) and 6-dimethylaminopurine (6-DMAP) were used to examine the effects of protein dephosphorylation on the control of mitosis in mammalian cells. Both 2-AP and 6-DMAP induced premature mitosis in hamster fibroblasts that were arrested in S phase. This response was characterized by changes in cell morphology, breakdown of the nuclear envelope, and premature chromosome condensation. Premature mitosis was followed by a return to interphase morphology and reformation of the nuclear envelope around decondensed and fragmented chromatin to form numerous micronuclei. The activity of both compounds was dependent upon new protein synthesis but not new RNA synthesis. 2-AP and 6-DMAP acted cooperatively with each other and with caffeine, suggesting a common mechanism of action. In exponentially growing cells, 2-AP and 6-DMAP did not induce premature mitosis but did increase the frequency of binucleated cells by blocking cytokinesis. These findings support a role for protein dephosphorylation in the control of mitosis and indicate that cell cycle perturbations can modify this regulation.