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Biomedical subjects

A S Alberts

Publications and source records attributed to A S Alberts.

At least 37 records · Page 2Linked to original sources
PubMed1994

Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3T3 fibroblasts.

We have examined the activity and phosphorylation state of the cyclic AMP (cAMP) response element binding factor (CREB) in intact NIH 3T3 cells following microinjection of expression plasmids encoding regulatory proteins of type 1 (PP1) and 2A (PP2A) serine/threonine-specific protein phosphatases. Changes in CREB phosphorylation in the injected cells were monitored by indirect immunofluorescence using an affinity-purified antiserum (Ab5322) which specifically recognizes CREB phosphorylated at Ser-133, and changes in transcriptional activity of CREB were monitored by expression of a reporter gene regulated by cAMP. cAMP-stimulated phosphorylation in NIH 3T3 cells is normally transient, and as expected, after stimulation of cells with cell-permeable cAMP analogs, the level of phosphorylated CREB was found to initially increase and then return to a basal level within 4 h. Microinjection of an expression vector encoding a constitutively active form of inhibitor 1 (I-1), a PP1-specific inhibitor, by itself resulted in an apparent increase in phosphorylated CREB in unstimulated cells. Moreover, injection of the I-1 vector resulted in the prolonged appearance of phosphorylated CREB in cells after cAMP stimulation. In contrast, injection of a plasmid encoding simian virus 40 small t antigen, which interacts with PP2A to inhibit its activity towards several phosphoprotein substrates, had no effect on the phosphorylation state of CREB in stimulated or unstimulated NIH 3T3 cells. Consistent with these results, injection of the I-1 expression vector activated expression from a coinjected CRE-lacZ reporter plasmid, indicating that the increased phosphorylation of CREB also activated its transcriptional activity. These results provide further evidence for a role of a PP1 as the primary protein (Ser/Thr) phosphatase regulating the dephosphorylation of Ser-133 and thereby limiting the transcriptional activity of CREB.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Simian virus 40 small t antigen cooperates with mitogen-activated kinases to stimulate AP-1 activity.

The simian virus 40 small tumor antigen (small t) specifically interacts with protein phosphatase type 2A (PP2A) in vivo and alters its catalytic activity in vitro. Among the substrates for PP2A in vitro are the activated forms of MEK and ERK kinases. Dephosphorylation of the activating phosphorylation sites on MEK and ERKs by PP2A in vitro results in a decrease in their respective kinase activities. Recently, it has been shown that overexpression of small t in CV-1 cells results in an inhibition of PP2A activity toward MEK and ERK2 and a constitutive upregulation of MEK and ERK2 activity. Previously, we have observed that overexpression of either ERK1, MEK1, or a constitutively active truncated form of c-Raf-1 (BXB) is insufficient to activate AP-1 in REF52 fibroblasts. We therefore examined whether overexpression of small t either alone or in conjunction with ERK1, MEK1, or BXB could activate AP-1. We found that coexpression of small t and either ERK1, MEK1, or BXB resulted in an increase in AP-1 activity, whereas expression of either small t or any of the kinases alone did not have any effect. Similarly, coexpression of small t and ERK1 activated serum response element-regulated promoters. Coexpression of kinase-deficient mutants of ERK1 and ERK2 inhibited the activation of AP-1 caused by expression of small t and either MEK1 or BXB. Coexpression of an interfering MEK, which inhibited AP-1 activation by small t and BXB, did not inhibit the activation of AP-1 caused by small t and ERK1. In contrast to REF52 cells, we observed that overexpression of either small or ERK1 alone in CV-1 cells was sufficient to stimulate AP-1 activity and that this stimulation was not enhanced by expression of small t and ERK1 together. These results show that the effects of small t on immediate-early gene expression depend on the cell type examined and suggest that the mitogen-activated protein kinase activation pathway is distinctly regulated in different cell types.

Animals↗
PubMed1993

Regulation of cell cycle progression and nuclear affinity of the retinoblastoma protein by protein phosphatases.

Decreased affinity of the retinoblastoma protein (RB) for the nuclear compartment has been correlated with cell cycle-dependent phosphorylation of the RB protein during the G1/S phase of the cell cycle. We examined the effects of microinjected protein-serine/threonine phosphatases types 1 (PP1) and 2A (PP2A) on nuclear association of RB monitored as the resistance of RB to extraction at the G1/S transition. Microinjection of PP1 into either the nucleus or the cytoplasm of cells synchronized in G1 increased the amount of RB that was resistant to extraction from the nucleus. Microinjection of PP2A, however, required direct injection into the nucleus to generate this effect. In addition, we found that nuclear injection of only the PP2A catalytic subunit (PP2AC) and not the complex containing the A and C subunits inhibited RB extraction. Microinjection of either PP1 or PP2A and the resultant increased affinity of RB for the nucleus corresponded with the inhibition of cell cycle progression into S phase. Injection of either phosphatase into cells that had entered S phase did not block DNA synthesis, suggesting that the effect of the injected phosphatases on cell cycle arrest was specific. In vitro biochemical studies with purified PP1 and PP2A showed that intact RB protein phosphorylated by cdc2 kinase served as a substrate for both protein phosphatases. Our results suggest that protein phosphatases may be important regulators of RB function and support the idea that cell cycle progression is regulated by the phosphorylation state of the RB protein.

Animals↗
PubMed1993

Signal transduction through the cAMP-dependent protein kinase.

Temporal cellular events responsible for hormonal activation of responses mediated by the cAMP-dependent protein kinase (PKA) have been studied in living cells. By selectively perturbing molecular function of Gs, the catalytic subunit of PKA (C), or the nuclear factor CREB, in cells through microinjection of inhibitory agents specific for these molecules or activated forms of these molecules, we have obtained evidence for a requirement for the function of each of these molecules in the hormonal stimulation of cAMP-regulated genes. Moreover, by introducing fluorescently labeled PKA subunits into these cells as molecular tracers, or by immunofluorescence of C subunit, we have observed biological translocation of C subunit from the cytoplasm to the nucleus during transcriptional activation and a quenching of this by the inhibitor molecule, PKI. The implications of these cellular and molecular events in the signal transduction of hormonal responses are discussed.

Animals↗
PubMed1993

The value of radiotherapy or chemotherapy after intubation for advanced esophageal carcinoma--a prospective randomized trial.

After intubation for advanced, inoperable squamous carcinoma of the oesophagus, a prospectively controlled randomized trial was done to investigate the effect of radiotherapy (41 patients) or mono-chemotherapy (40 patients) or no further treatment (46 patients). Treatment had no significant effect on either palliation or survival (p < or = 0.7) and did not alter the natural history of the disease.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1993

Rapid transcriptional assay for the expression of two distinct reporter genes by microinjection.

We have developed a technique in which gene expression from multiple reporter plasmids introduced by needle microinjection can be monitored simultaneously in individual cells by double-label indirect immunofluorescence. With constitutively active viral promoters, expression from lacZ or chloramphenicol acetyl transferase (CAT) reporter genes can be detected within as little as 30 min after injection. Expression from such strong promoters reaches a maximum level after about 2 hr. In place of the constitutive promoter, promoters containing different enhancer elements respond as expected to different stimuli, allowing for the comparison of two defined transcriptional control elements in living cells. Reporter expression can be analyzed temporally and can be compared to expression of endogenous genes. This technique is complementary to transfection and allows for the targeted analysis of expression in specific cells, for example, in a mixed cell population, and for the analysis of expression in cells that are available only in small numbers.

Animals↗
PubMed1993

Protein phosphatase 2A potentiates activity of promoters containing AP-1-binding elements.

The involvement of serine/threonine protein phosphatases in signaling pathways which modulate the activity of the transcription factor AP-1 was examined. Purified protein phosphatase types 1 (PP1) and 2A (PP2A) were microinjected into cell lines containing stably transfected lacZ marker genes under the control of an enhancer recognized by AP-1. Microinjection of PP2A potentiated serum-stimulated beta-galactosidase expression from the AP-1-regulated promoter. Similarly, transient expression of the PP2A catalytic subunit with c-Jun resulted in a synergistic transactivation of an AP-1-regulated reporter gene. PP2A, but not PP1, potentiated serum-induced c-Jun expression, which has been previously shown to be autoregulated by AP-1 itself. Consistent with these results, PP2A dephosphorylated c-Jun on negative regulatory sites in vitro, suggesting one possible direct mechanism for the effects of PP2A on AP-1 activity. Microinjection of PP2A had no effect on cyclic AMP (cAMP)-induced expression of a reporter gene containing a cAMP-regulated promoter, while PP1 injection abolished cAMP-induced gene expression. Taken together, these results suggest a specific role for PP2A in signal transduction pathways that regulate AP-1 activity and c-Jun expression.

Animals↗
PubMed1992

A phase II study of 5-fluorouracil and leucovorin in advanced carcinoma of the esophagus.

Thirty-five patients with advanced epidermoid carcinoma of the esophagus were treated with 5-fluorouracil (5-FU) 425 mg/m2 and leucovorin 20 mg/m2, day 1-5 every 28 days. Six patients had a partial response (95% confidence limit, 7-35%) with a median response duration of 32 weeks. The median survival time of the patients on study was 14 weeks. The toxicity was acceptable, with only two patients experiencing severe hematologic toxicity and one patient experiencing severe nausea and vomiting. The addition of leucovorin at this dose level in this population of patients with advanced disease does not appear to enhance the activity of 5-FU for patients with squamous cell cancer of the esophagus. Since only a small percentage of patients experienced significant toxicity, a higher response rate could be achieved in patients treated with the maximally tolerated dose.

Adult↗
PubMed1991

Metastatic breast cancer--age has a significant effect on survival.

The data on 217 elderly (aged greater than or equal to 65 years) and 209 middle-aged postmenopausal patients with metastatic breast cancer treated in the Department of Medical Oncology, University of Pretoria, from 1976 to 1985 were analysed to determine the effect of age on survival. When considered as a group, the elderly have a more favourable prognosis (median survival 20.3 months) than the middle-aged (median survival 15.54 months) (P = 0.0457). Multivariate age subset analysis (taking into account all other major prognostic factors) reveal a distinct bimodal pattern. The median survival of patients aged 45-54 years is 21.2 months and decreases to 16.2 months for patients aged 55-64 years (P = 0.08; Cox model). The median survival improves again to 24.6 months for patients aged 64-74 years (P = 0.0001; Cox model), followed by an apparent but non-significant decrease to 17.1 months in the very old (aged 75-84 years) (P = 0.52; Cox model). The more favourable prognosis in the elderly dictates effective non-toxic treatment.

Age Factors↗
PubMed1991

Differential regulation of jun family gene expression by the tumor promoter okadaic acid.

Phosphorylation events are major regulatory mechanisms of signal transduction pathways that control cell growth and differentiation. The potential involvement of serine/threonine-specific phosphoprotein phosphatases in pathways that regulate gene expression was analyzed. By use of okadaic acid, an inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A), we present evidence that expression of distinct members of the jun family of genes, c-jun, junB, and junD, are regulated differentially by serine/threonine-specific phosphoprotein phosphatases. Treatment of cells with okadaic acid induces the expression of junB, and to a lesser extent c-jun, but has only a marginal effect on junD expression. This induction involves transcriptional as well as post-transcriptional mechanisms. An analysis of defined elements in different promoters suggests that serine/threonine phosphoprotein phosphatases are involved in the regulation of the c-jun and the collagenase 12-O-tetradecanoyl phorbol-13-acetate (TPA) response element (TRE) as well as the c-fos serum response element (SRE). Since inhibition of PP-1 and PP-2A leads to increased proto-oncogene expression, our results further support the view that certain protein phosphatases might act as negative regulators of growth.

3T3 Cells↗
PubMed1991

Factors influencing prognosis in elderly patients with primary breast cancer.

A retrospective analysis was performed on 260 elderly and 294 middle-aged patients with primary breast cancer. Cox regression analysis showed that the classic prognostic factors, such as negative lymph node status, small tumour size and positive oestrogen receptor (ER) status, were favourable indicators of survival and were similar in both the elderly and the middle-aged. When the elderly and middle-aged were considered together, older age (favourable) and the presence of association disease (detrimental) were additional significant prognostic factors. It is concluded that a physically fit elderly woman in the present study had a more favourable survival outlook than a younger counterpart with similar tumor size, lymph node and ER status and should be given the best available treatment based on tumour characteristics and not on age alone.

Age Factors↗
PubMed1990

A phase II study of idarubicin and prednisone in multiple myeloma.

Twenty-one patients with multiple myeloma were treated with idarubicin 45 mg/m2 orally day 1 and prednisone 60 mg/m2 day 1-4 every three weeks. Moderate to severe gastrointestinal and hematopoietic toxicity were observed. Twelve of the twenty-one patients had relapsed on prior treatment. Of these, 2 patients responded. Two patients had primary resistant disease, neither responded. Seven patients had received no prior treatment, three responded. Idarubicin and prednisone have modest activity in refractory myeloma, with short duration of response and severe toxicity.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1990

Construction of mammalian cell lines with indicator genes driven by regulated promoters.

The regulation of gene expression is a central feature of cell growth and differentiation. An important question is how information received at the cell surface is ultimately transmitted to the nucleus and how it causes changes in the pattern of gene expression. To study the molecular basis of this process, we have established cell lines carrying marker genes under the control of known regulatory promoter elements. These cell lines are being used to investigate the effects of activating normal cellular second messenger systems or of microinjecting proteins hypothesized to function in signal transduction, such as oncogene products or subunits of enzymes.

Animals↗
PubMed1990

"Idiopathic thrombocytopenic purpura-like syndrome" treated with interferon in a patient with lung cancer.

A case of autoimmune thrombocytopenia associated with lung cancer is presented. The thrombocytopenia occurred 2 years before the diagnosis of lung cancer. Previous treatments include high doses of corticosteroids and splenectomy. The patient was managed with interferon, which resulted in an increase of the platelet count with control of the hemorrhagic manifestations with this syndrome.

Carcinoma, Small Cell↗