PAP modulations in Daudi cells and Molt-3 cells treated with etoposide are mutually associated with morphological evidence of apoptosis.

Daudi (B-cell line) and Molt-3 (T-cell line) cells provide a model for the study of apoptosis, the induction of which is often accompanied by concominant modulations of proteins involved in mRNA maturation. One of these proteins is poly(A) polymerase (PAP), which is responsible for mRNA cleavage and polyadenylation. A number of recent reports also suggest involvement of mRNA maturation and stability in the induction of specific pathways of cell apoptosis. In this study we identified PAP activity levels and isoform modulations in two different cell lines (Daudi and Molt-3) and related them to DNA fragmentation (a hallmark of apoptosis) and cell cycle phase specificity in terms of the temporal sequence of events and the time that elapsed between administration of the apoptosis inducer (the widely used anticancer drug etoposide) and the observed effects. Treatment of both cell lines with 20 microg/mL etoposide induced apoptosis after four hours in Molt-3 cells and only after 24 hours in Daudi cells, as revealed by two independent methods. In Daudi cells the PAP activity levels and isoforms were downregulated prior to deltapsim reduction, DNA fragmentation and the morphological changes of the nucleus, whereas in Molt-3 cells no PAP activity and isoform modulations were observed prior to the early hallmarks of apoptosis.

C-MYC and IGF-II mRNA-binding protein (CRD-BP/IMP-1) in benign and malignant mesenchymal tumors.

Mouse coding region determinant-binding (mCRD-BP) and human IGF-II mRNA-binding 1 (hIMP-1) proteins are orthologous mRNA-binding proteins that recognize c-myc and IGF-II mRNA, respectively, and regulate their expression posttranscriptionally. Here, we confirm that human CRD-BP/IMP-1 binds to c-myc mRNA and that it is predominantly expressed in fetal tissues. Moreover, hCRD-BP/IMP-1 expression was detected in cell lines of neoplastic origin and in selected primary tumors. In a series of 33 malignant and 10 benign mesenchymal tumors, 73% and 40%, respectively, were found to express hCRD-BP/IMP-1. In particular, expression was significant in 14 Ewing's sarcomas, all of which were positive. The data suggest that hCRD-BP/IMP-1 plays a role in abnormal cell proliferation in mesenchymal tumors.

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.

Polyadenylate polymerase enzymatic activity in mammary tumor cytosols: A new independent prognostic marker in primary breast cancer.

Polyadenylate polymerase (PAP) is one of the enzymes involved in the formation of the polyadenylate tail of the 3' end of mRNA. High levels of PAP activity were associated with rapidly proliferating cells. Here we evaluate the prognostic value of PAP activity in breast cancer patients. PAP specific activity values were measured by a highly sensitive assay in the tumor cytosols of 228 women with primary breast cancer. The median follow-up period was 58 months. PAP specific activity values ranged from 2.1-39.4 units/mg protein in the breast tumor cytosols, and the activity was correlated with the level of expression of the antigen. An optimal cutoff value of 5.5 units/mg extracted protein was first defined by statistical analysis. PAP status was then compared with other established prognostic factors in terms of relapse-free survival (RFS) and overall survival (OS). PAP activity levels had a tendency to increase with tumor-node-metastasis (TNM) stage and were higher in node-positive patients. Evaluation of the prognostic value of PAP was performed using univariate and multivariate analyses. Univariate analysis showed that PAP-positive patients had a less favorable prognosis for both RFS (relative risk (RR) = 2.35; P < 0.001] and OS (RR = 3.15; P < 0.001). PAP significantly added to the prognostic power for RFS (RR = 2.51; P = 0.0012) and OS (RR = 4.21; P < 0.001) in multivariate analysis, whereas patient age, tumor size, and nodal and ER status remained independent factors for predicting survival. When only node-negative patients were examined, PAP was found to be an independent factor for predicting RFS (RR = 3.68; P = 0.0032) and OS (RR = 4.81; P < 0.001). PAP did not appear to have a prognostic significance for node-positive patients. PAP is a new prognostic factor for early recurrence and death in breast cancer patients. Our results suggest that PAP may be used as an independent unfavorable prognostic factor in node-negative breast cancer patients because there were no significant associations between PAP and the other prognostic indicators evaluated in this group of patients.

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.

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.

Early 5-fluorouracil-induced changes of poly(A) polymerase in HeLa and WISH cells.

5-Fluorouracil (5-FU), a drug with numerous mechanisms of action which has a long-term suppressive effect on human cancer cell proliferation, mediates both partial dephosphorylation and inactivation of poly(A) polymerase (PAP) [EC. 2.7.7.19] as detected by immunoblotting analysis and non-specific enzyme assay, respectively, in human carcinoma HeLa and diploid WISH cells at a concentration of 100 microM. When the same experiment is done in the presence of phosphatase inhibitors, 5-FU-induced partial PAP dephosphorylation is abolished. Moreover, a cell type-modulated, differential response of HeLa cells (5-FU chemosensitive cells) versus WISH cells (drug-resistant diploid cells) is observed. These results suggest that 5-FU induces early direct or indirect changes in the structure and function of PAP and may regulate pre-mRNA cleavage-polyadenylation.

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.

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.

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.

[Age-dependent changes in mRNA transport (nucleus-cytoplasm)]. / Alternsabhängige Veränderungen des mRNA-Transports (Kern-Zytoplasma).

Transport of mRNA from nucleus to cytoplasm is an ATP-dependent process which occurs strictly vectorially. Because the mRNA is structurally bound during transport, mRNA transport is a "solid-state" process consisting of i) mRNA release from the nuclear matrix, ii) mRNA translocation through the nuclear pore, and iii) cytoskeletal binding. We identified and purified the following components involved in the translocation step: i) the nuclear envelope (NE) nucleoside triphosphatase (NTPase) which is stimulated by the 3'poly(A) tail of mRNA, ii) the poly(A)-recognizing mRNA carrier, iii) the NE protein kinase, and iv) the NE phosphatase. In addition, we found that an RNA helicase activity is present in NE, which also may be involved in RNA transport. Our results show that, besides poly(A), also double-stranded RNA structures may modulate RNA export. The amount of mRNA released from nuclei markedly decreases with age. Evidence is presented that this age-dependent change is caused by an impairment of polyadenylation of mRNA, hnRNA processing, release of mRNA from nuclear matrix, and translocations of mRNA from nuclear to cytoplasmic compartment (decrease in activities of NE NTPase, protein kinase, and phosphatase; decrease in poly(A)-binding affinity of mRNA carrier).

Patterns of adenosine deaminase, ecto-5'-nucleotidase, poly(A)polymerase and surface light chain expression in chronic lymphocytic leukemias.

The levels of activity of three enzymes have been measured in the circulating malignant lymphocytes of 47 patients with B chronic lymphocytic leukemia (CLL). These were the purine degradative enzymes, adenosine deaminase (ADA) and ecto-5'-nucleotidase (5'NT) and the enzyme responsible for the polyadenylation of mRNA, poly(A) polymerase. The patterns of activity of the above enzymes and the expression of surface immunoglobulin light chains were examined. A heterogeneity in the specific activity of the enzymes was observed which could not be attributed to variations of the percentage of B lymphocytes. A positive correlation was found between ADA and poly(A)polymerase activity (r = 0.383, p less than 0.01). Furthermore, the expression of immunoglobulin light chain phenotype was inversely related to 5'NT specific activity; CLL cases in which less than 20% of the cells expressed lambda chain phenotype, presented 5'NT specific activity of 16.7 +/- 3.3 (S.E.) nmol/h/10(6) cells, whereas in CLL cases with more than 20% of the cells expressing this phenotype the enzyme specific activity was 4.8 +/- 1.6 (S.E.) nmol/h/10(6) cells (p less than 0.02). These findings suggest that the simultaneous determination of enzymatic activities and immunological markers, might be useful in defining subsets in CLL and the subsequent clinical treatment.

Relation between the activity of poly(A)polymerase and the levels of protein synthesis in the lymphocytes of patients with chronic lymphocytic leukemia.

The activity levels of the enzyme poly(A)polymerase and the levels of protein synthesis primed by endogenous messenger RNA (mRNA) as well as polyuridylic acid, poly(U) directed polyphenylalanine synthesis, were determined in lymphocytic extracts from 17 patients with chronic lymphocytic leukemia of the B cell type. The enzyme activity values have not been found to correlate with the poly(U)-protein synthesis, whereas a positive linear correlation has been established between the activity levels of poly(A)polymerase and the endogenous mRNA-primed protein synthesis (r = 0.735, p less than 0.01). This difference between exogenously and endogenously primed protein synthesis in concern with poly(A)polymerase is discussed.

Soluble cAMP-independent protein kinase from human spleen.

A protein kinase (EC 2.7.1.37) was purified 2000-fold, from the soluble protein fraction of human spleen cells, using ion-exchange chromatography, ammonium sulfate fractionation, and gel filtration. This rapid procedure yielded 30% of the initial activity and an enzyme preparation with specific activity of 62 nmol min-1 mg-1 of protein. On the basis of disc gel electrophoresis in sodium dodecyl sulfate acrylamide gels and isoelectric focusing the enzyme preparation appears homogeneous and to consist of one polypeptide with a molecular weight of 43,000 and having a pI of 7.1. The purified enzyme activity is cyclic AMP and cGMP independent phosphorylates both alpha-casein and phosvitin, and uses Mg2+ ATP and Mg2+ GTP as phosphate donors, exhibiting an apparent Km of 2.0 and 6.6 X 10(-5)m, respectively. Furthermore, the enzyme activity is strongly inhibited by heparin (K50 = 0.1 micrograms/ml). These catalytic properties are characteristic of the enzyme casein kinase II, as described in several eukaryotic cells.

A colorimetric assay for the determination of 5'-nucleotidase activity.

A rapid, simple, quantitative and sensitive assay for the determination of 5'-nucleotidase has been developed. The method can be applied to both soluble and membrane bound forms of the leukocyte enzyme. Enzyme activity is determined by colorimetric estimation of NH3 released from adenosine, the product of 5'-nucleotidase activity in the presence of adenosine deaminase. The assay may be performed in microtitre plates and read with an automatic multiscan spectrophotometer. Thus it can be applied to a large number of samples for routine medical and research purposes.

Increase in the levels of activity of polyadenylic acid-metabolizing enzymes following phytohaemagglutinin stimulation of human lymphocytes.

Increased levels of soluble activity of all three enzymes involved in polyadenylic acid metabolism were measured in PHA-stimulated versus normal lymphocytes. Poly(A)-polymerase and poly(A)-exonuclease values increased significantly (from 25.7 +/- 4.2 (S.E.M.) to 53.5 +/- 10.6 (S.E.M.), and from 334.6 +/- 33.2 (S.E.M.) to 653.2 +/- 53.4 (S.E.M.) respectively), while a moderate increase was observed in poly(A)-endonuclease (from 299.2 +/- 33.8 (S.E.M.) to 403.0 +/- 77.1 (S.E.M.). The above differences persisted after two fractionations of the crude cell extracts by ion exchange chromatography and molecular sieving, and could not be attributed to the competitive action of all three enzymes in the untreated extracts. Fractionation of the extracts of resting and stimulated cells on Sephadex G-75 revealed two molecular forms of poly(A)-polymerase activity.