Replicative senescence is characterized by irreversible growth arrest and has been defined by four genetic complementation groups. One of these groups is associated with the predominance of ...underphosphorylated, growth-suppressive retinoblastoma tumor suppressor protein (pRb). Although certain members of the cyclin-dependent kinase (cdk)/cyclin family, some of which phosphorylate pRb, are underexpressed in senescent cells, others are expressed but inactive. This lack of cdk activity and arrest in the G1 phase of the cell cycle is likely attributable to the induction upon senescence of the G1-S cdk/cyclin inhibitors p21 (WAF1/CIP1/Sdi) and p16INK4. In fact, in early presenescent normal diploid fibroblasts in which p21 is inactivated, senescence is bypassed or postponed. Moreover, in senescent cells in which p53 function was inhibited, DNA synthesis was reinitiated, an effect likely attributable, in part, to the dependence of p21 expression on p53. We report here that the apparent inactivation of p21 in senescent human fibroblasts through the introduction of inhibitory alpha-p21 antibodies causes these cells to reenter the S-phase of the cell cycle. The disruption of p21 activity affects the p21-Rb-E2F pathway in that the expression of genes transcriptionally regulated by E2F, such as cyclin A and cdc2, were found to be up-regulated in injected cells. No evidence of cell division was observed. This suggests that p21 plays an important role in the maintenance of senescence and in the inhibition of S-phase progression, but inhibition of p21 activity is insufficient to permit cells to complete the cell cycle.
Using an E. coli expression-vector system we have efficiently produced, purified, and characterized the full-length, nonfused, protooncogenic and oncogenic (T-24) forms of the human H-ras gene ...product. These purified ras proteins have been introduced by microinjection into a variety of somatic cells in an effort to examine their function. Within several hours after injection of the oncogenic form of the human H-ras protein into quiescent cells, we observe dramatic morphological changes followed by transient proliferation of the cells. In contrast, microinjection of the normal, protooncogenic form of the ras protein at the same level appears to have only little effect on the cells. Additional experiments indicate that the effect of the ras protein requires entry into the cells, is temporary, is inhibited by cycloheximide or actinomycin D, and is seen only in established cell lines. This experimental approach demonstrates that the bacterially derived and purified human H-ras proteins retain their ability to function when put back into mammalian cells and furthermore, provides a novel assay for transformation induced in established cells by the human H-ras oncogene protein.
Transcriptional regulation of eukaryotic genes by cyclic AMP requires a cAMP-dependent protein kinase (A kinase). Two hypotheses have been proposed to explain how the holoenzyme of the A kinase ...induces transcription. The regulatory subunits of the A kinase, which bind cAMP and DNA, and have amino-acid homology with the Escherichia coli catabolite activator protein could directly stimulate gene expression. Alternatively, phosphorylation by the catalytic subunits could induce transcription by activating proteins involved in gene transcription. To distinguish between these models, we microinjected purified preparations of the catalytic and regulatory subunits of A kinase into tissue culture cells and monitored expression of a stably integrated fusion gene containing a cAMP-responsive human promoter fused to a bacterial reporter gene, or of the endogenous c-fos gene. The catalytic subunit stimulated expression of these genes, whereas the regulatory subunit did not. These results indicate that the catalytic subunit of A kinase is sufficient to induce expression of two cAMP-responsive genes, without increasing levels of cAMP.
Wounding of tissue induces cellular responses that ultimately result in wound repair. Studies in tissue culture model systems indicate that these responses include induction of AP-1 regulated genes, ...cell migration and mitogenesis which are also characteristic of cellular responses to growth factors. Investigations have identified cellular ras proteins as critical components of growth factor-stimulated signal transduction pathways, however their role in the wounding response is less clear. Investigation of the potential involvement of c-Ras in this process utilized quiescent living bovine corneal endothelium cells (BCE) which were microinjected with ras dominant interfering mutant protein (N17) and subsequently stimulated by mechanical wounding. Analysis of these cells demonstrated that microinjection of dominant-interfering ras protein, but not control proteins, inhibited the wounding response as evidenced by diminished Fos expression, lack of cell migration and a block in DNA synthesis.
Adenylyl cyclase type 6 (AC6) and the β
1 adrenergic receptor (β
1AR) are pivotal proteins in transmembrane βAR-signaling in cardiac myocytes. Increased expression of AC6 has beneficial effects on ...the heart, but increased β
1AR expression has marked deleterious effects. Why do these two elements of the βAR pathway have such different effects? Using adenovirus-mediated gene transfer of the two transgenes in neonatal rat cardiac myocytes, we assessed cellular distribution and performed selected biochemical assays. β
1AR was found predominantly in the plasma membrane. In contrast, AC6 was found in the plasma membrane but also was associated with the nuclear envelope, sarcoplasmic reticulum, mitochondria, and cytoplasm. Increased β
1AR, but not AC6, increased follistatin expression, p38 phosphorylation, phosphatidylserine translocation to the PM, and apoptosis. In contrast, increased AC6, but not β
1AR, inhibited PHLPP2 activity, activated PI3K and Akt, and increased p70S6 kinase phosphorylation and Bcl-2 expression; apoptosis was unchanged. The distribution of AC6 to multiple cellular compartments appears to enable interactions with other proteins (e.g., PHLPP2) and activates cardioprotective signaling (PI3K/Akt). In contrast, β
1AR, confined to the plasma membrane, increased phosphatidylserine translocation and apoptosis. These data provide a potential underlying mechanism for the beneficial vs deleterious effects of these two related βAR-signaling elements.
Adenylyl cyclase type 6 (AC6) and the ß
1
adrenergic receptor (ß
1
AR) are pivotal proteins in transmembrane ßAR-signaling in cardiac myocytes. Increased expression of AC6 has beneficial effects on ...the heart, but increased ß
1
AR expression has marked deleterious effects. Why do these two elements of the ßAR pathway have such different effects? Using adenovirus-mediated gene transfer of the two transgenes in neonatal rat cardiac myocytes, we assessed cellular distribution and performed selected biochemical assays. ß
1
AR was found predominantly in the plasma membrane. In contrast, AC6 was found in the plasma membrane but also was associated with the nuclear envelope, sarcoplasmic reticulum, mitochondria, and cytoplasm. Increased ß
1
AR, but not AC6, increased follistatin expression, p38 phosphorylation, phosphatidylserine translocation to the PM, and apoptosis. In contrast, increased AC6, but not ß
1
AR, inhibited PHLPP2 activity, activated PI3K and Akt, and increased p70S6 kinase phosphorylation and Bcl-2 expression; apoptosis was unchanged. The distribution of AC6 to multiple cellular compartments appears to enable interactions with other proteins (eg, PHLPP2) and activates cardioprotective signaling (PI3K/Akt). In contrast, ß
1
AR, confined to the plasma membrane, increased phosphatidylserine translocation and apoptosis. These data provide a potential underlying mechanism for the beneficial vs deleterious effects of these two related ßAR-signaling elements.
We have investigated the role of the proto-oncogene HRas in cardiac cell growth and hypertrophy. By direct needle microinjection
of activated Ras protein into primary neonatal rat ventricular cardiac ...myocytes, we find that, unlike many other cell types,
Ras does not induce DNA synthesis in these cells. However, injection of activated Ras does induce expression of both the c-Fos
and atrial natriuretic factor (ANF) genes. Expression of both these genes is associated with the hypertrophic response in
ventricular myocytes suggesting that Ras is involved in the hypertrophic signalling pathway. Ras injection also causes morphological
changes in the cells so that they increase in profile and show changes in the organization of the contractile apparatus. Further
support for a role for Ras in the hypertrophic response was obtained from studies showing that activated Ras stimulates ANF
promoter activity in transient transfection assays. We also show that a dominant interfering Ras mutant inhibits the hypertrophic
stimulation of the ANF promoter by phenylephrine, indicating a role for Ras in the hypertrophic effect of an alpha-adrenergic
agonist.
PDF
