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Biomedical subjects

M Havredaki

Publications and source records attributed to M Havredaki.

At least 19 recordsLinked to original sources
PubMed2001

Drug action on poly(A) polymerase activity and isoforms during U937 cell apoptosis.

The enzyme poly(A) polymerase (PAP; EC 2.7.7.19) catalyzes the polyadenylation of mRNAs. It's activity levels and isoforms vary within the cell cycle (31) and apoptosis (34). The direct effect of most anticancer drugs is cell damage (DNA and RNA synthesis inhibition, DNA breaks and/or cell cycle aberrations), which then triggers signaling pathways that activate apoptosis and eventually lead to regulated cell death. The experiments described here concern the chemotherapeutic agents, interferon (IFN) and 5-fluorouracil (5-FU), and their action on U937 cells, alone or in various combinations, resulting in the commitment of cell apoptosis, as observed by the appearance of DNA fragmentation. Furthermore, examination of U937 cell apoptotic trend in parallel with PAP activity measurements and isoforms detection by immunoblotting, revealed both partial enzyme inactivation and dephosphorylation, in particular after the combined drug action of 5-FU and IFN on U937 cells. Our work on chemotherapeutic drug action at the level of mRNA polyadenylation may contribute to new insights into the mechanism of cell apoptosis, as well as provide information on mRNA poly(A) tail formation, removal and function.

Apoptosis↗
PubMed1999

The polyadenylation inhibitor cordycepin (3'dA) causes a decline in c-MYC mRNA levels without affecting c-MYC protein levels.

Study of the distribution of the poly(A) tail length of c-myc mRNA in several cell lines revealed a distinct, prevailing population with short poly(A) tails, derived through sequential deadenylation. To elucidate the possible in vivo function of this distinct short tailed c-myc mRNA population, the polyadenylation inhibitor cordycepin was used. This resulted in a decline in steady state c-myc mRNA levels with the remaining messenger mostly oligoadenylated. However, c-MYC proteins did not follow the reduction of the c-myc mRNA. On the other hand, in cells exposed to physiological agents known to downregulate c-myc expression, the reduction of mRNA steady state levels, was reflected upon c-MYC protein levels. The dissociation between c-myc mRNA and protein levels caused by cordycepin was not due to the stabilization of the c-MYC proteins and was not an indiscriminate effect since in the presence of cordycepin, c-fos mRNA and protein levels concomitantly declined. Our data indicate that under these conditions, a long poly(A) tail is not instrumental for c-myc mRNA translation and furthermore, the discrepancy in the steady state of c-myc mRNA level: c-MYC protein ratio between control cells and cells treated with cordycepin indicates that c-myc mRNA is subjected to translational control.

Deoxyadenosines↗
PubMed1998

Dephosphorylation, proteolysis, and reduced activity of poly(A) polymerase associated with U937 cell apoptosis.

The apoptotic trend of the widely used cell lines HL-60, U937, HeLa, Molt-3, and K562 has been found to be accompanied and reversibly related with Poly(A) polymerase (PAP; EC 2.7.7.19) activity levels. Moreover, variations in the pattern of multiple enzyme forms are revealed, being most prominent in apoptosis-prone cell lines, HL-60 and U937. Furthermore, in heat-shocked or nutrient-deprived apoptotic U937 Percoll-fractionated subpopulations, PAP lower mobility phosphorylated forms of 106 and 100 kDa as well as enzyme activity were progressively reduced along with the appearance of higher than 80 kDa mobility species. The kinetics of these alterations (dephosphorylation, proteolysis, and activity) coincided with the appearance of DNA fragmentation. In fact, PAP dephosphorylation appears to precede the appearance of DNA fragmentation. In addition, inhibition of PAP dephosphorylation, proteolysis, and decrease in its activity were tightly coupled with the concomitant prevention of apoptosis. This novel finding yields information on a possible involvement of PAP in cell commitment and execution to apoptosis.

Apoptosis↗
PubMed1997

Interferon-inhibiting activities in sera of patients with lung cancer.

A differential pattern of sera exhibiting interferon-inhibiting activity in cases of histologically confirmed lung cancer patients have been observed. The sera distribution was dependent on the clinical entity and the cell line specificity used measuring IFN-inhibition. Although either small cell lung cancer (SCLC) or non small cell lung cancer (NSCLC) sera exhibiting in a high percentage (80 and 75% respectively) such an activity, in any of the 3 lines tested, the ratio of sera exerting inhibition in each cell line was different. Such cell specific systems could be of prognostic/predicting value for effective therapeutic schedules of specific clinical entities.

Aged↗
PubMed1996

In vivo generation of 3' and 5' truncated species in the process of c-myc mRNA decay.

It has been demonstrated that the half-life of c-myc mRNA is modulated in response to physiological agents. The elucidation of the decay process and the identification of the critical steps in the in vivo c-myc mRNA degradation pathway can be approached by following the fate of c-myc mRNA under the influence of such factors. IFN-alpha was the factor used to modulate c-myc mRNA half-life in HeLa 1C5 cells, a stable clone derived from HeLa cells. This cell line carries multiple copies of the c-myc gene, under the control of the dexamethasone inducible mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Exposure of HeLa 1C5 cells to IFN-alpha resulted in a further 2-fold increase over the dexamethasone-induced c-myc mRNA. However, the c-myc mRNA in IFN-alpha treated cells was less stable than that in the control cells. RNase H mapping of the 3' untranslated region of c-myc mRNA revealed, in addition to the full length mRNA, three smaller fragments. These fragments were proven to be truncated, non-adenylated c-myc mRNA species generated in vivo. Exposure of HeLa 1C5 cells to Interferon-alpha before induction with dexamethasone resulted in the enhanced presence of these intermediates. RNase H analysis of c-myc mRNA after actinomycin D chase revealed that deadenylation led to the formation of a relatively more stable oligoadenylated c-myc mRNA population which did not appear to be precursor to the truncated intermediates. The detection of truncated 3' end c-myc mRNA adenylated fragments as well, implies that the c-myc mRNA degradation process may follow an alternative pathway possibly involving endonucleolytic cleavage.

Animals↗
PubMed1996

Biochemical and immunological identification and enrichment of poly(A) polymerase from human thymus.

Human thymus poly(A) polymerase (EC 2.7.7.19) activity has been investigated using poly(A) and oligo(A) as initiators. All obtained fractions reveal more than one polypeptide as detected by immunoblotting after SDS-PAGE. In addition to the homogeneously purified (Tsiapalis et al., J Biol Chem 250: 4486-4496, 1975 and Wahle, J Biol Chem 266: 3131-3139, 1991), about 60 kDa polypeptide, a larger polypeptide, about 80 kDa, that comigrates in the region of poly(A) polymerase activity was detected, enriched and partially characterized; it appears having similar size with bovine poly(A) polymerase cloned in E. coli. Polyclonal antiserum produced against recombinant bovine poly(A) polymerase reacts more efficiently with the about 80 kDa polypeptide upon immunoblotting, and can precipitate the poly(A) polymerase activity. This enzyme form, from human tissue, is novel in terms of size and may reflect intact or physiological form of poly(A) polymerase in human thymus, and supports and substantiates recent reports on the enzyme from other sources.

Animals↗
PubMed1993

Interferon inhibitors and/or inactivators in sera from patients with neoplasias.

Measurements of interferons, their inhibitors and/or inactivators and tumor necrosis factor-like activities in sera from patients with neoplasias were performed. Interferon activity could not be detected in 96% of the sera samples examined. In contrast, both interferon inhibitors and/or inactivators and tumor necrosis factor-like activities were found in a high proportion (25-100%) of sera from cancer patients but not in sera from normal individuals.

Cytotoxicity, Immunologic↗
PubMed1993

Interferon caused alterations of human cell response to bleomycin in vitro.

HeLa cells were exposed to recombinant interferon-alpha (rIFN-alpha) in combination with bleomycin (BLM) concentrations known to exert single as well as double-strand breakages of cellular DNA. rIFN-alpha is added before or/and after cell treatment with BLM. The sensitivity of HeLa cells to BLM or/and rIFN was assessed from the number and the size of cell colonies formed. rIFN-alpha alone at concentrations of 250 IU/ml had no effect on the development of HeLa cell colonies, in combination with BLM, rIFN-alpha increased the already marked inhibition of HeLa cell colony formation caused by 1 microgram/ml BLM. At BLM concentrations of 5 and 10 micrograms/ml the inhibition of HeLa cell colony formation was almost complete and no overlapped effect of BLM and rIFN-alpha can be distinguished. However, rIFN-alpha afforded protection of HeLa cells colony formation when it was added before BLM treatment or it was present continuously thereafter.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1987

Polyadenylate polymerase activity in stationary and growing cell cultures.

Soluble polyadenylic acid (poly(A] polymerase content of stationary and growing cell populations from a variety of cell lines was determined. Cell populations from stationary cultures presented poly(A) polymerase values with a mean of 31 +/- 12 enzyme units/mg protein. The mean value for growing cell populations were 62 +/- 18 enzyme units per mg protein. A statistically significant difference was found between stationary and growing cell populations from the variety of cell lines examined (p less than 0.1). The observed differences in poly(A) polymerase levels persisted after fractionation of the crude extracts and revealed two molecular forms of enzyme activity with a net charge difference in stationary and growing cell cultures.

Adenosine Monophosphate↗
PubMed1986

Circulating interferon-like activity in inflammatory bowel disease.

Inflammatory bowel diseases (IBD) has been characterized as having an autoimmune background. We studied the presence of immune interferon in the sera of a small number of patients with IBD. Antiviral activity was detectable in the sera of our patients with well-controlled IBD but it was mainly weak-positive (50% inhibition of virus CPE in a dilution of 1:10 per 0.2 ml of serum). The antiviral activity varied according to the sensitivity of the cell line used. The human amnion cells Wish (81%) and the human embryonic Intestine-407 (73%) were more sensitive.

Antiviral Agents↗
PubMed1986

Virus-inhibiting factor in primary and metastatic carcinoma of the liver.

Sera from patients with hepatoma, metastatic liver disease, cirrhosis of the liver, and benign diseases of the digestive tract have been tested for a virus-inhibiting factor. Serum samples exerting complete inhibition of virus CPE in a dilution of at least 1:10 per 0.2 ml are considered positive, and those exerting 50% inhibition of CPE are regarded as weakly positive. An apparently positive antiviral activity was noted in the sera of patients with hepatoma (53.3%) and metastatic liver disease (46.6%). Patients with cirrhosis of the liver showed positivity in one of ten sera; no positive antiviral activity could be found in the sera of patients with benign diseases of the digestive tract. The percentage of weakly positive response was 33.3% and 13.3% for hepatoma and metastatic liver disease, respectively, and 10.0% for the benign diseases and 20.0% for the cirrhotic sera. The antiviral activity was more prominent in the embryonic foreskin fibroblasts and to a lesser degree in the bovine epithelial cells NBL-1.

Antiviral Agents↗
PubMed1985

Variations of interferon inactivators and/or inhibitors in human serum and their relationship to interferon therapy.

Anti-interferon (IFN) activities of normal human sera and sera from cases suspected of viral infection were determined. Of the normal sera tested (57), 65% presented anti-IFN activities against IFN-alpha and/or -beta preparations; 61% against IFN-beta and 32% against IFN-alpha. Of the sera from suspected virus-infected individuals (44), 82% presented anti-IFN activity to IFN-beta and 57% to IFN-alpha with the majority of sera (91%) exerting anti-IFN activities to IFN-alpha and/or -beta preparation. The observed variations of anti-IFN alpha and beta activities of human sera appear to be reproducible and should be taken into consideration in the clinical application of IFN.

Humans↗
PubMed1975

Virus susceptibility of mouse hemopoietic cells in vitro: inhibition of granulocyte-macrophage precursor cells by Newcastle disease virus.

Normal mouse bone marrow cells were exposed to encephalomyocarditis virus (EMC), reovirus type 3 (REO3), influenza virus (FLU), and Newcastle disease virus (NDV) then assayed for granulocyte-macrophage precursor cells by the technique of colony formation in agar. Exposure to EMC, REO3, and FLU caused a slight but variable loss of colony-forming potential, whereas exposure to NDV caused a very marked loss. NDV acted directly on the cells, not indirectly through release of colony-inhibiting factors or destruction of colony-stimulating factor. Experiments with NDV inactivated by heat, ether, or ultraviolet irradiation indicated that colony inhibition was associated with fully infective virus, even though some of the inactivated preparations had retained full hemagglutinin, neuraminidase, or hemolytic activity.

Animals↗
PubMedDate not supplied

Poly(A) polymerase specifically implicated in the mechanism of chemotherapeutic drug action during cell apoptosis.

It has recently been established that most anticancer drugs act through the mechanism of apoptosis. It has also been clinically confirmed that drug combinations are more effective than single drugs and various chemotherapeutic strategies have therefore been developed. The experiments described here concern the induction of apoptosis with dimethylsulfoxide (DMSO), a substance with multiple activity especially as an inducer of differentiation, and interferon (IFN), a cytokine well known for its antiviral and antineoplastic effects; they are used alone or in combination. Apoptosis may be regulated at all levels of gene expression including the addition of the poly(A) tail to the 3' end of mRNAs. Poly(A) polymerase (PAP) [EC.2.7.7.19], the enzyme that catalyzes the addition of the poly(A) tail to mRNAs, changes in the process of development, differentiation, transformation and apoptosis. In the present study the induction of HeLa cells to apoptosis (65%) with a DMSO/rIFN-alpha combination resulted in pronounced PAP dephosphorylation and activity reduction. HeLa cells induced to apoptosis (35%) with DMSO gave lower levels of PAP dephosphorylation and reduction of activity and cells induced to apoptosis (18%) with rIFN-alpha gave only limited PAP dephosphorylation and reduction of activity. The implications of these observations for chemotherapeutic drug action at the level of mRNA polyadenylation point to the possible use of PAP as a biological marker for the evaluation of this action.

Antineoplastic Agents↗
PubMedDate not supplied

Anticancer drug action on poly(A) polymerase activity and isoforms during HeLa and WISH cell apoptosis.

Poly(A) polymerase (PAP; EC 2.7.7.19) catalyzes mRNA polyadenylation. Its activity and isoform levels vary during cell cycle transformation and apoptosis. It has become widely accepted that cell death after DNA damage by anticancer agents is primarily the result of apoptosis and that cells able to evade apoptosis will be resistant to cell killing. The therapeutic agents interferon (IFN), 5-fluorouracil (5-FU) and tamoxifen (Tam) with different mechanisms of action mediate both partial dephosphorylation and inactivation of PAP, detected by immunoblotting analysis and PAP enzyme assay, respectively. We examined the apoptotic tendencies of HeLa and WISH cell lines caused by one of the drugs used, 5-FU. The trend in the cells examined, observed by DAPI and/or DNA fragmentation assay, was found to be accompanied by and reversibly related to PAP activity levels and PAP lower mobility phosphorylated forms of 106 and 100 kDa isoforms. Moreover, a cell type-modulated, differential response of HeLa (chemosensitive cells) versus WISH (drug-resistant diploid cells) has been revealed. This finding yields information on the possible use of PAP as a tumor marker involved in cell commitment and/or induction of apoptosis and may help to improve our understanding of tumor cell sensitivity to anticancer agents.

Amnion↗