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Biomedical subjects

B T Gjertsen

Publications and source records attributed to B T Gjertsen.

At least 37 records · Page 2Linked to original sources
PubMed1999

Ultrarapid caspase-3 dependent apoptosis induction by serine/threonine phosphatase inhibitors.

The protein phosphatase (PP) inhibitors nodularin and microcystin-LR induced apoptosis with unprecedented rapidity, more than 50% of primary hepatocytes showing extensive surface budding and shrinkage of cytoplasm and nucleoplasm within 2 min. The apoptosis was retarded by the general caspase inhibitor Z-VAD.fmk. To circumvent the inefficient uptake of microcystin and nodularin into nonhepatocytes, toxins were microinjected into 293 cells, Swiss 3T3 fibroblasts, promyelocytic IPC-81 cells, and NRK cells. All cells started to undergo budding typical of apoptosis within 0.5 - 3 min after injection. This was accompanied by cytoplasmic and nuclear shrinkage and externalization of phosphatidylserine. Overexpression of Bcl-2 did not delay apoptosis. Apoptosis induction was slower and Z-VAD.fmk independent in caspase-3 deficient MCF-7 cells. MCF-7 cells stably transfected with caspase-3 showed a more rapid and Z-VAD.fmk dependent apoptotic response to nodularin. Rapid apoptosis induction required inhibition of both PP1 and PP2A, and the apoptosis was preceded by increased phosphorylation of several proteins, including myosin light chain. The protein phosphorylation occurred even in the presence of apoptosis-blocking concentrations of Z-VAD.fmk, indicating that it occurred upstream of caspase activation. It is suggested that phosphatase-inhibiting toxins can induce caspase-3 dependent apoptosis in an ultrarapid manner by altering protein phosphorylation.

3T3 Cells↗
PubMed1999

Implications of cell death regulation in the pathogenesis and treatment of prostate cancer.

Although the phenomenon of programed cell death, or apoptosis, has been recognized for many years, interest in the clinical implications of apoptotic cell death has not been widely apparent until recently. It is now established that the products of at least some oncogenes and tumor suppressor genes are able to regulate the rates of apoptosis as well as proliferation in tumor cell populations. In fact, it appears that evasion of deletion via apoptotic cell death may be a requisite event in the development of many malignant neoplasms. Molecular alterations that occur at high frequency in the pathogenesis of prostate cancer often involve genes implicated in the regulation of cell death. There is now ample reason to speculate that the susceptibility of individual malignant neoplasms to undergo apoptosis in response to a given therapeutic intervention may be a useful parameter in predicting therapeutic response. This realization has profound implications with respect to the design and implementation of treatment strategies for prostate cancer based on the biology of this disease.

Apoptosis↗
PubMed1998

Clobetasol propionate followed by calcipotriol is superior to calcipotriol alone in topical treatment of psoriasis.

BACKGROUND: Although potent, topical corticosteroids offer effective and rapid healing of psoriatic lesions. Their long term use is limited because of the risk of side effects. Calcipotriol is safe for long-term treatment, but its initial efficacy is lower than with topical corticosteroids. OBJECTIVES: To investigate whether 2 weeks of treatment with clobetasol propionate 0.05% ointment bd followed by 4 weeks of treatment with calcipotriol 50 microg/g bd would offer therapeutic advantages over 6 weeks of continuous treatment with calcipotriol. METHODS: Forty-nine patients with moderate to severe plaque psoriasis were recruited from five centres in Norway. In a randomised, double-blind, right- versus left-side comparison, ointments were applied to two symmetrically-located areas. RESULTS: Two weeks of treatment with clobetasol propionate produced a significantly greater decrease in total symptom score (combined scores of erythema, induration and scaling) than calcipotriol treatment (P < 0.0001). This improvement on the clobetasol propionate-treated side of the body was maintained throughout a subsequent 4-week treatment period when calcipotriol was applied to both sides of the body (P < 0.0001). The superiority of the clobetasol propionate followed by calcipotriol treatment was maintained during a 4-week, treatment-free, observation period. Treatments were well tolerated with no rebound effect. CONCLUSIONS: Clobetasol propionate ointment bd for 2 weeks followed by treatment with calcipotriol ointment bd for 4 weeks was superior to calcipotriol ointment alone in the treatment of plaque psoriasis.

Administration, Topical↗
PubMed1997

Fas/APO-1(CD95)-induced apoptosis of primary hepatocytes is inhibited by cAMP.

Fas/APO-1(CD-95) activation induced rapid apoptotic cell death of primary rat hepatocytes in suspension culture. Activators of cAMP-dependent protein kinase (glucagon and N6-benzoyl-cAMP) protected against apoptosis, whereas the specific cAMP-kinase inhibitor (Rp)-8-Br-cAMPS enhanced Fas-induced death. The latter observation indicated that even the basal cAMP level may provide partial protection against Fas-induced hepatocyte apoptosis. Two-dimensional gel electrophoresis revealed decreased phosphorylation of several proteins in Fas-activated cells. Most of these dephosphorylations were attenuated or not observed in cells simultaneously stimulated by anti-Fas and cAMP, indicating a tight correlation between the dephosphorylations and death. Elevation of cAMP rescued the cells not only from the Fas-induced morphological changes and dephosphorylation, but also from functional deterioration. Whereas cells treated with anti-Fas alone quickly lost plating efficiency, hepatocytes co-treated with glucagon retained their ability to adhere and spread on a collagen substratum.

Animals↗
PubMed1997

Gap junctions and growth control in liver regeneration and in isolated rat hepatocytes.

The hepatocytes in the mature normal liver are tightly coupled through gap junctions, except during compensatory hyperplasia (regeneration) after partial hepatectomy when the gap junctions become down-regulated. The significance of this down-regulation has been a long-standing enigma. The present study of hepatocytes in primary culture and in the regenerating liver aimed at defining the relationship, if any, between hepatocyte gap junctional communication and proliferation. Gap junctional down-regulation in the regenerating liver appeared to be a specific phenomenon because desmosomes and the surface contact area between neighboring hepatocytes remained constant. All agents and conditions (dexamethasone in vivo; dexamethasone, cyclic adenosine monophosphate, serum, and high cell density in vitro) delaying gap junctional down-regulation also increased the lag before the cells reached competence to enter S phase. This raised the possibility that hepatocyte DNA replication was inhibited through preservation of gap junctions. However, we disproved this assumption by showing that the DNA replication (more specifically the G1/S transition rate constant) was inhibited even in hepatocytes completely devoid of gap junctional communication. The teleological advantage of linking gap junctional down-regulation to hepatocyte G1 progression therefore may not be to trigger DNA replication but to ensure that proliferating hepatocytes and hepatocytes responsible for liver-specific metabolic functions maintain separate pools of metabolites and signaling molecules.

Animals↗
PubMed1997

Inherited mutations in PTEN that are associated with breast cancer, cowden disease, and juvenile polyposis.

PTEN, a protein tyrosine phosphatase with homology to tensin, is a tumor-suppressor gene on chromosome 10q23. Somatic mutations in PTEN occur in multiple tumors, most markedly glioblastomas. Germ-line mutations in PTEN are responsible for Cowden disease (CD), a rare autosomal dominant multiple-hamartoma syndrome. PTEN was sequenced from constitutional DNA from 25 families. Germ-line PTEN mutations were detected in all of five families with both breast cancer and CD, in one family with juvenile polyposis syndrome, and in one of four families with breast and thyroid tumors. In this last case, signs of CD were subtle and were diagnosed only in the context of mutation analysis. PTEN mutations were not detected in 13 families at high risk of breast and/or ovarian cancer. No PTEN-coding-sequence polymorphisms were detected in 70 independent chromosomes. Seven PTEN germ-line mutations occurred, five nonsense and two missense mutations, in six of nine PTEN exons. The wild-type PTEN allele was lost from renal, uterine, breast, and thyroid tumors from a single patient. Loss of PTEN expression was an early event, reflected in loss of the wild-type allele in DNA from normal tissue adjacent to the breast and thyroid tumors. In RNA from normal tissues from three families, mutant transcripts appeared unstable. Germ-line PTEN mutations predispose to breast cancer in association with CD, although the signs of CD may be subtle.

Adenoma↗
PubMed1995

Novel (Rp)-cAMPS analogs as tools for inhibition of cAMP-kinase in cell culture. Basal cAMP-kinase activity modulates interleukin-1 beta action.

Novel (Rp)-cAMPS analogs differed widely in ability to antagonize cAMP activation of pure cAMP-dependent protein kinase I and II and to antagonize actions of cAMP on gene expression, shape change, apoptosis, DNA replication, and protein phosphorylation in intact cells. These differences were related to different abilities of the analogs to stabilize the holoenzyme form relative to the dissociated form of cAMP kinase type I and II. (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were the most potent cAMP antagonists for isolated type I kinase and for cells expressing mostly type I kinase, like IPC-81 leukemia cells, fibroblasts transfected with type I regulatory subunit (RI), and primary hepatocytes. It is proposed that (Rp)-8-Br-cAMPS or (Rp)-8-Cl-cAMPS should replace (Rp)-cAMPS as the first line cAMP antagonist, particularly for studies in cells expressing predominantly type I kinase. The phosphorylation of endogenous hepatocyte proteins was affected oppositely by (Rp)-8-Br-cAMPS and increased cAMP, indicating that (Rp)-8-Br-cAMPS inhibited basal cAMP-kinase activity. The inhibition of basal kinase activity was accompanied by enhanced DNA replication, an effect which could be reproduced by microinjected mutant cAMP-subresponsive RI. It is concluded that the basal cAMP-kinase activity exerts a tonic inhibition of hepatocyte replication. (Rp)-8-Br-cAMPS and microinjected RI also desensitized hepatocytes toward inhibition of DNA synthesis by interleukin-1 beta. This indicates that basal cAMP-kinase activity can have a permissive role for the action of another (interleukin-1 beta) signaling pathway.

3T3 Cells↗
PubMed1995

Sensitive and rapid detection of beta-galactosidase expression in intact cells by microinjection of fluorescent substrate.

Bacterial beta-galactosidase, coded by lacZ, is a widely used reporter for studies of transcriptional activity of eukaryotic promoters at the single cell level. Unfortunately, current detection methods, like X-gal cytochemistry, are slow, have suboptimal sensitivity, and are incompatible with cell survival. By a novel approach based on microinjection into cells of the fluorogenic substrate 5-chloromethylfluorescein di-beta-D-galactopyranoside lacZ gene expression was detected without affecting cell viability or proliferative capacity. The method was far more sensitive than the conventional X-gal cytochemistry in all cell systems tested (primary hepatocytes, fibroblasts, and glioma cells). Results were obtained within seconds to minutes after injection, and cells remained fluorescent for hours.

Animals↗
PubMed1995

8-Chloro-cAMP induces apoptotic cell death in a human mammary carcinoma cell (MCF-7) line.

8-Cl-cAMP and 8-NH2-cAMP induced MCF-7 cell death. The type(s) of cell death were studied in more detail and compared with the cell death type (apoptosis) induced by okadaic acid, an inhibitor of serine/threonine phosphatases. By morphological criteria dying cells showed loss of cell-cell interactions and microvilli, condensation of nuclear chromatin and segregation of cytoplasmic organelles. By in situ nick end-labelling, using digoxigenin-conjugated dUTP as probe, a large fraction of 8-Cl-cAMP, 8-NH2-cAMP and 8-Cl-adenosine-exposed cells stained positively in the advanced stages of death. In the early phase of chromatin condensation the cells stained negatively. Specific (internucleosomal) DNA fragmentation was not observed. The MCF-7 cell death induced by 8-Cl-cAMP and 8-NH2-cAMP was not mediated by activation of the cAMP kinase since more stable cAMP analogues (8-CPT-cAMP and N6-benzoyl-cAMP) or forskolin failed to induce death. Furthermore, 8-Cl-cAMP action was counteracted by adenosine deaminase and 3-isobutyl-1-methylxanthine, and mimicked by 8-Cl-adenosine, a major metabolite of 8-Cl-cAMP. It is concluded that 8-Cl- and 8-NH2-cAMP can induce morphological and biochemical effects resembling apoptotic cell death in MCF-7 cells through their conversion into potent cytotoxic metabolite(s).

1-Methyl-3-isobutylxanthine↗
PubMed1995

Fine mapping of 28S rRNA sites specifically cleaved in cells undergoing apoptosis.

Bona fide apoptosis in rat and human leukemia cells, rat thymocytes, and bovine endothelial cells was accompanied by limited and specific cleavage of polysome-associated and monosome-associated 28S rRNA, with 18S rRNA being spared. Specific 28S rRNA cleavage was observed in all instances of apoptotic death accompanied by internucleosomal DNA fragmentation, with cleavage of 28S rRNA and of DNA being linked temporally. This indicates that 28S rRNA fragmentation may be as general a feature of apoptosis as internucleosomal DNA fragmentation and that concerted specific cleavage of intra- and extranuclear polynucleotides occurs in apoptosis. Apoptosis-associated cleavage sites were mapped to the 28S rRNA divergent domains D2, D6 (endothelial cells), and D8. The D2 cuts occurred in hairpin loop junctions considered to be buried in the intact ribosome, suggesting that this rRNA region becomes a target for RNase attack in apoptotic cells. D8 was cleaved in two exposed UU(U) sequences in bulge loops. Treatment with agents causing necrotic cell death or aging of cell lysates failed to produce any detectable limited D2 cleavage but did produce a more generalized cleavage in the D8 region. Of potential functional interest was the finding that the primary cuts in D2 exactly flanked a 0.3-kb hypervariable subdomain (D2c), allowing excision of the latter. The implication of hypervariable rRNA domains in apoptosis represents the first association of any functional process with these enigmatic parts of the ribosomes.

Animals↗
PubMed1994

Cleaved intracellular plasminogen activator inhibitor 2 in human myeloleukaemia cells is a marker of apoptosis.

The proteolytic modification of plasminogen activator inhibitor 2 (PAI-2) was studied during apoptosis in the human promyelocytic leukaemic NB4 cell line during treatment with the phosphatase inhibitors okadaic acid and calyculin A as well as the protein synthesis inhibitor cycloheximide. The apoptic type of cell death was ascertained by morphological and biochemical criteria. In cell homogenates PAI-2 was probed by [125I]urokinase plasminogen activator (uPA) and detected as a sodium dodecyl sulphate-stable M(r) 80,000 complex after reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. During apoptosis a smaller (M(r) 70,000) uPA-PAI-2 complex was consistently detected. The modification was in the PAI-2 moiety, as the [125I]uPA tracer could be extracted in its intact form from the complex. Thus the cleaved PAI-2 isoform is a biochemical marker of apoptosis in the promyelocytic NB4 cell line. The modified PAI-2 isoform was also detected in homogenates made from purified human mononuclear leukaemic cells aspirated from the bone marrow of patients suffering from acute and chronic myeloid leukaemia.

Apoptosis↗
PubMed1994

Multiple apoptotic death types triggered through activation of separate pathways by cAMP and inhibitors of protein phosphatases in one (IPC leukemia) cell line.

The protein phosphatase inhibitors okadaic acid and calyculin A at moderate concentrations induced three types of apoptotic promyelocytic leukemia cell death, distinct with respect to ultrastructure and polynucleotide fragmentation. Calyculin A at higher concentrations (> 50 nM) induced a non-apoptotic death type with high ATP and pronounced micromitochondriosis. This suggests that protein phosphorylation pathways are involved in the triggering of several death pathways. Activation of the cAMP kinase induced yet another apoptotic death type, preferentially affecting cells in S-phase. In fact, cAMP acted in two ways to stop IPC promyelocyte proliferation: (1) block in late G1 (preventing new cells from entering DNA replication); and (2) induction of apoptosis in S-phase. cAMP and phosphatase inhibitors acted via distinct pathways. The inhibitors suppressed cAMP-induced death, but only at concentrations high enough to commit the cells to alternative, less conspicuous death types. The tumor-promoting activity of okadaic acid and calyculin A may therefore not be by protection against apoptosis. DNA fragmentation correlated with the novel feature of limited 28 S rRNA cleavage, suggesting co-ordinated polynucleotide cleavage, possibly directed against illegitimate polynucleotides, in some apoptotic death types.

Animals↗
PubMed1993

Exogenous substrate stimulates autodephosphorylation of cyclic-AMP-dependent protein kinase II.

The autophosphorylated regulatory subunit (32P-RII) of cyclic-AMP-dependent protein kinase II was efficiently dephosphorylated by its C subunit in the absence of added ADP, provided that Mg/ATP and a standard protein kinase peptide substrate were present. This raises the possibility that autodephosphorylation could be significant in the intact cell. Only the cyclic-AMP-complexed free form of 32P-RII was efficiently dephosphorylated, indicating that the autodephosphorylation was intermolecular. Autodephosphorylation of 32P-RII in the presence of MgATP and kemptide occurred with formation of [gamma-32P]ATP, suggesting transfer of 32P of phospho-RII to a transient C*(MgADP) complex formed during the forward kinase reaction with peptide as substrate. Autodephosphorylation promoted by phosphorylation of exogenous substrates could operate also for other kinases conforming to a mechanism where MgADP remains bound to the active site after the other product (phosphorylated substrate) has left the catalytic complex.

Adenosine Diphosphate↗
PubMed1993

[Pemphigus vulgaris and benign cicatricial mucous membrane pemphigoid in the upper respiratory tract and esophagus].

Pemphigus vulgaris and benign cicatricial membrane pemphigoid are both autoimmune, blistering, dermatologic diseases characterised clinically by tense bullae on skin or on mucous membranes. Both diseases are rare, but very serious, associated with a high death rate (pemphigus) or high morbidity with cicatricial mucosal lesions (pemphigoid) if untreated. These diseases are discussed and two case stories mentioned where the primary focus was in the upper aerodigestive tract, which is very seldom. The otolaryngologist can make an important contribution to the early recognition, diagnosis, and management of these diseases. The biopsy must undergo immunofluorescence examination.

Diagnosis, Differential↗
PubMed1993

Antiapoptotic effect of heterozygously expressed mutant RI (Ala336-->Asp) subunit of cAMP kinase I in a rat leukemia cell line.

cAMP induced rapid apoptosis (> 90% cell death in 6 h) of non-growth-arrested rat leukemia IPC-81 cells. A cell clone selected for cAMP resistance had a normally functioning apoptotic machinery whose triggering required about 30-fold higher cellular cAMP than in the parent cells. The cAMP subresponsiveness was due to a heterozygous point mutation (Ala336-->Asp) in the RI subunit of cAMP-dependent protein kinase I. In fact, apoptosis correlated with intracellular cAMP binding to the subresponsive RI. The mutated alanine is invariantly present in cyclic nucleotide kinases, but of unknown function. The mutation decreased the cAMP affinity to site B by increasing the cAMP dissociation rate 500x. The ability of site B to discriminate adenine-modified cAMP analogues was affected, suggesting that Ala336 faced the adenine moiety of cAMP. That the heterozygously expressed RID336 was a dominant suppressor of apoptosis was explained by a higher expression of R than C subunits in the mutant cells by preferential expression of the mutant form of RI, and by the ability of mutant RI to exert dominant negative control of activation of wild type cAMP kinase at moderate cAMP levels. Apoptosis was induced at a similar cAMP level in cells treated with cholera toxin or other cAMP elevating agents, indicating that cAMP kinase was essential for toxin action.

Alanine↗
PubMed1993

Microinjected catalytic subunit of cAMP-dependent protein kinase induces apoptosis in myeloid leukemia (IPC-81) cells.

Rat IPC-81 promyelocytic leukemia cells responded to cAMP analog by undergoing apoptotic cell death both when anchored to fibronectin and when free in the medium. The protein kinase C stimulator 12-O-tetradecanoylphorbol 13-acetate enhanced the anchoring to substratum without impeding cAMP-induced cell death. The immobilized cells could be microinjected. This made it possible to study the effect on apoptosis of microinjected catalytic (C alpha) and regulatory (RI alpha D199) subunits of cAMP-dependent protein kinase as well as of phosphatase inhibitors. Microinjection of C alpha reproduced the morphological effects of cAMP, including nuclear fragmentation. RI alpha D199 blocked the effect of C alpha. Injection of microcystin-LR, which inhibits protein phosphatases 1 and 2A, led to pronounced apoptoid changes of the leukemia cells, but failed to produce nuclear fragmentation. Microinjection of peptide inhibitors ("inhibitor 1" and "inhibitor 2") specific for phosphatase 1 had no effect on cell morphology. The failure of the phosphatase inhibitors to reproduce completely the effect of the C subunit underscores the specificity of action of the latter.

Animals↗