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M E Smulson

Publications and source records attributed to M E Smulson.

At least 19 recordsLinked to original sources
PubMed1998

Induction of apoptosis in proliferating human endothelial cells by the tumor-specific antiangiogenesis agent combretastatin A-4.

The antiangiogenic, tubulin-binding drug combretastatin A-4 exhibits a selective toxicity for proliferating endothelial cells in vitro and induces vascular shutdown in tumor models in vivo. The mechanism of combretastatin A-4 cytotoxicity has now been investigated with cultured proliferating human umbilical vein endothelial cells by examining various markers of apoptosis. Incubation of cells with 0.1 mM combretastatin A-4 induced the conversion (first detected after 6 h) of the CPP32 proenzyme to active caspase-3, a cysteine protease that plays an important role in apoptosis in many cell types; the drug also increased caspase-3 activity. Another early event observed was the binding of annexin V to 50% of the cells 8 h after drug treatment. Internucleosomal DNA fragmentation, another hallmark of apoptosis, was detected in cells incubated with 0.1 mM combretastatin A-4 for 24 h. Staining with Hoechst 33258 revealed that about 75% of cells exhibited a nuclear morphology characteristic of apoptosis after incubation with drug for 24 h. Incubation of cells for up to 8 h with combretastatin A-4 did not induce the release of lactate dehydrogenase or increase the uptake of propidium iodide, both indicators of membrane integrity. These results indicate that the selective cytotoxic effect of combretastatin A-4 is mediated by the induction of apoptosis rather than by necrosis and may provide an enhanced clinical strategy in cancer chemotherapy with this new agent.

Animals↗
PubMed1998

The E7 protein of human papillomavirus type 16 sensitizes primary human keratinocytes to apoptosis.

The 'high risk' human papillomaviruses are associated with the development of anogenital carcinomas and their E6 and E7 genes possess immortalizing and transforming functions in several in vitro culture systems. Recently the E6 gene has also been shown to enhance the apoptosis of human mammary epithelial cells. To determine the apoptotic activity of these oncogenes in the natural host cell, we infected genital keratinocytes with retroviruses expressing either HPV-16 E6, E7, or both the E6 and E7 (E6/7) genes. Apoptosis was quantitated under normal growth conditions or when induced by tumor necrosis factor alpha/cycloheximide or sulfur mustard. In contrast to previous findings with mammary epithelial cells, the E6 gene did not significantly augment either spontaneous or induced apoptosis. E6 also did not suppress apoptosis in normal keratinocytes (despite dramatically reducing their p53 levels), suggesting that p53-independent events mediated this effect. In contrast, E7 increased both spontaneous and induced apoptosis as well as the cellular levels of p53 and p21 protein. Interestingly, co-expression of E6 abrogated E7-facilitated apoptosis by tumor necrosis factor alpha nearly completely, but had only a minor protective effect on sulfur mustard induced apoptosis in these cells, demonstrating at least in part the p53-dependence and -independence of these two apoptotic pathways. Finally, our results indicate that the apoptosis of normal and E7-expressing keratinocytes is differentially affected by E6 expression and that E7, when unaccompanied by E6, sensitizes keratinocytes to apoptosis.

Apoptosis↗
PubMed1998

Irreversible binding of poly(ADP)ribose polymerase cleavage product to DNA ends revealed by atomic force microscopy: possible role in apoptosis.

During apoptosis, DNA undergoes fragmentation and caspase-3 cleaves poly(ADP-ribose) polymerase (PARP) into both a 24-kDa fragment containing the DNA binding domain and an 89-kDa fragment containing the catalytic and automodification domains. Atomic force microscopy revealed that recombinant full-length PARP bound to plasmid DNA fragments and linked them into chainlike structures. Automodification of PARP in the presence of NAD+ resulted in its dissociation from the DNA fragments, which, nevertheless, remained physically aligned. A recombinant 28-kDa fragment of PARP containing the DNA binding domain but lacking the automodification domain irreversibly bound to and linked DNA fragments in the absence or presence of NAD+. Identical results were obtained on incubation of internucleosomal DNA fragments from apoptotic cells with the products of cleavage of recombinant PARP by purified caspase-3. The 24-kDa product of PARP cleavage by caspase-3 may contribute to the irreversibility of apoptosis by blocking the access of DNA repair enzymes to DNA strand breaks.

Animals↗
PubMed1998

Regulation of the expression or recruitment of components of the DNA synthesome by poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP) is a component of the multiprotein DNA replication complex (MRC, DNA synthesome) that catalyzes replication of viral DNA in vitro. PARP poly(ADP-ribosyl)ates 15 of the approximately 40 proteins of the MRC, including DNA polymerase alpha (DNA pol alpha), DNA topoisomerase I (topo I), and proliferating-cell nuclear antigen (PCNA). Although about equal amounts of MRC-complexed and free forms of PCNA were detected by immunoblot analysis of HeLa cell extracts, only the complexed form was poly(ADP-ribosyl)ated, suggesting that poly(ADP-ribosyl)ation of PCNA may regulate its function within the MRC. NAD inhibited the activity of DNA pol delta in the MRC in a dose-dependent manner, whereas the PARP inhibitor, 3-AB, reversed this inhibitory effect. The roles of PARP in modulating the composition and enzyme activities of the DNA synthesome were further investigated by characterizing the complex purified from 3T3-L1 cells before and 24 h after induction of a round of DNA replication required for differentiation of these cells; at the latter time point, approximately 95% of the cells are in S phase and exhibit a transient peak of PARP expression. The MRC was also purified from similarly treated 3T3-L1 cells depleted of PARP by antisense RNA expression; these cells do not undergo DNA replication nor terminal differentiation. Both PARP protein and activity and essentially all of the DNA pol alpha and delta activities exclusively cosedimented with the MRC fractions from S phase control cells, and were not detected in the MRC fractions from PARP-antisense or uninduced control cells. Immunoblot analysis further revealed that, although PCNA and topo I were present in total extracts from both control and PARP-antisense cells, they were present in the MRC fraction only from induced control cells, indicating that PARP may play a role in their assembly into an active DNA synthesome. In contrast, expression of DNA pol alpha, DNA primase, and RPA was down-regulated in PARP-antisense cells, suggesting that PARP may be involved in the expression of these proteins. Depletion of PARP also prevented induction of the expression of the transcription factor E2F-1, which positively regulates transcription of the DNA pol alpha and PCNA genes; thus, PARP may be necessary for expression of these genes when quiescent cells are stimulated to proliferate.

3T3 Cells↗
PubMed1998

Transient poly(ADP-ribosyl)ation of nuclear proteins and role of poly(ADP-ribose) polymerase in the early stages of apoptosis.

A transient burst of poly(ADP-ribosyl)ation of nuclear proteins occurs early, prior to commitment to death, in human osteosarcoma cells undergoing apoptosis, followed by caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP). The generality of this early burst of poly(ADP-ribosyl)ation has now been investigated with human HL-60 cells, mouse 3T3-L1, and immortalized fibroblasts derived from wild-type mice. The effects of eliminating this early transient modification of nuclear proteins by depletion of PARP protein either by antisense RNA expression or by gene disruption on various morphological and biochemical markers of apoptosis were then examined. Marked caspase-3-like PARP cleavage activity, proteolytic processing of CPP32 to its active form, internucleosomal DNA fragmentation, and nuclear morphological changes associated with apoptosis were induced in control 3T3-L1 cells treated for 24 h with anti-Fas and cycloheximide but not in PARP-depleted 3T3-L1 antisense cells exposed to these inducers. Similar results were obtained with control and PARP-depleted human Jurkat T cells. Whereas immortalized PARP +/+ fibroblasts showed the early burst of poly(ADP-ribosyl)ation and a rapid apoptotic response when exposed to anti-Fas and cycloheximide, PARP -/- fibroblasts exhibited neither the early poly (ADP-ribosyl)ation nor any of the biochemical or morphological changes characteristic of apoptosis when similarly treated. Stable transfection of PARP -/- fibroblasts with wild-type PARP rendered the cells sensitive to Fas-mediated apoptosis. These results suggest that PARP and poly(ADP-ribosyl)ation may trigger key steps in the apoptotic program. Subsequent degradation of PARP by caspase-3-like proteases may prevent depletion of NAD and ATP or release certain nuclear proteins from poly(ADP-ribosyl)ation-induced inhibition, both of which might be required for late stages of apoptosis.

3T3 Cells↗
PubMed1998

Sphingosine 1-phosphate inhibits activation of caspases that cleave poly(ADP-ribose) polymerase and lamins during Fas- and ceramide-mediated apoptosis in Jurkat T lymphocytes.

Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, sphingosine 1-phosphate (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (1996) Nature 381, 800-803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of sphingosine kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7.

Apoptosis↗
PubMed1998

Sulfur mustard induces markers of terminal differentiation and apoptosis in keratinocytes via a Ca2+-calmodulin and caspase-dependent pathway.

Sulfur mustard (SM) induces vesication via poorly understood pathways. The blisters that are formed result primarily from the detachment of the epidermis from the dermis at the level of the basement membrane. In addition, there is toxicity to the basal cells, although no careful study has been performed to determine the precise mode of cell death biochemically. We describe here two potential mechanisms by which SM causes basal cell death and detachment: namely, induction of terminal differentiation and apoptosis. In the presence of 100 microM SM, terminal differentiation was rapidly induced in primary human keratinocytes that included the expression of the differentiation-specific markers K1 and K10 and the cross-linking of the cornified envelope precursor protein involucrin. The expression of the attachment protein, fibronectin, was also reduced in a time- and dose-dependent fashion. Features common to both differentiation and apoptosis were also induced in 100 microM SM, including the rapid induction of p53 and the reduction of Bcl-2. At higher concentrations of SM (i.e., 300 microM), formation of the characteristic nucleosome-sized DNA ladders, TUNEL-positive staining of cells, activation of the cysteine protease caspase-3/apopain, and cleavage of the death substrate poly(ADP-ribose) polymerase, were observed both in vivo and in vitro. Both the differentiation and the apoptotic processes appeared to be calmodulin dependent, because the calmodulin inhibitor W-7 blocked the expression of the differentiation-specific markers, as well as the apoptotic response, in a concentration-dependent fashion. In addition, the intracellular Ca2+ chelator, BAPTA-AM, blocked the differentiation response and attenuated the apoptotic response. These results suggest a strategy for designing inhibitors of SM vesication via the Ca2+-calmodulin or caspase-3/PARP pathway.

Apoptosis↗
PubMed1996

The expression of poly(ADP-ribose) polymerase during differentiation-linked DNA replication reveals that it is a component of the multiprotein DNA replication complex.

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable to complete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified replicative complexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein replication complex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or modulates component replicative enzymes or factors in the complex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.

3T3 Cells↗
PubMed1995

Requirement for the expression of poly(ADP-ribose) polymerase during the early stages of differentiation of 3T3-L1 preadipocytes, as studied by antisense RNA induction.

Poly(ADP-ribose) polymerase (PADPRP) is biologically significant in the rejoining of DNA strand breaks. Post confluent cultures of 3T3-L1 preadipocytes showed marked increases in PADPRP protein and activity when the cells were induced to differentiate into adipocytes. When this increase in PADPRP expression was prevented in stably transfected 3T3-L1 cells by induction of PADPRP antisense RNA synthesis, the cells did not differentiate nor undergo the two or three rounds of DNA replication that are required for initiation of the differentiation process. 3T3-L1 cells expressing PADPRP antisense RNA under differentiation conditions were easily detached from plates and in some cases eventually died. When newly expressed PADPRP protein and DNA synthesis was assessed in cells at zero time or at 24 h after induction of differentiation by incorporation of bromodeoxyuridine or [3H]thymidine into DNA, significant incorporation was shown to occur in control cells after 24 h, but not in antisense cells. Furthermore, during the first 24 h, the co-immunoprecipitation of PADPRP and DNA polymerase alpha was observed in control cells, whereas no such complex formation was noted in the induced antisense cells, nor in uninduced control cells.

3T3 Cells↗
PubMed1995

Model systems for the study of the role of PADPRP in essential biological processes.

The nuclear enzyme poly(ADP-ribose) polymerase (PADPRP) is implicated in a number of cellular processes, including DNA repair, replication, and differentiation. We have been using several model systems to examine these potential roles of PADPRP. A human keratinocyte model system has been developed in which stable lines of epidermal cells contain an inducible construct harboring the antisense cDNA to PADPRP. When PADPRP antisense RNA is induced in culture, endogenous PADPRP mRNA, protein, and enzymatic activity is lowered, and the pattern of poly(ADP) ribosylation in response to alkylating agents is altered. When keratinocyte clones containing the antisense construct or empty vector alone were grafted onto nude mice, they formed histologically normal human skin. The PADPRP antisense construct was also inducible in vivo by the topical application of dexamethasone to the reconstituted epidermis, as determined by in situ hybridization. In addition, poly(ADP-ribose) polymer could be induced and detected in vivo following the topical application of a DNA alkylating agent to the grafted transfected skin layers. Thus, a model system has been developed in which the levels of PADPRP can be selectively manipulated in human keratinocytes in cell culture, and potentially in reconstituted epidermis as these keratinocyte lines can be grafted to nude mice, whereupon they form a histologically and immunocytochemically normal human epidermis. Another system that we have been utilizing to examine the role of PADPRP in proliferation and differentiation is stable lines of mouse preadipocytes that contain an inducible antisense PADPRP RNA. Similar to the keratinocyte system, these cells can inducibly express antisense PADPRP RNA, and subsequently lower levels of endogenous PADPRP.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1995

Engineered human skin model using poly(ADP-ribose) polymerase antisense expression shows a reduced response to DNA damage.

Poly(ADP-ribose) polymerase (PADPRP) modifies nuclear proteins in response to DNA-damaging agents. The principal organ subject to exposure to many of these agents is the skin. To understand the role of PADPRP in the maintenance of the epidermis, a model system has been developed in which we have selectively lowered the levels of this enzyme by the use of induced expression of antisense RNA. Human keratinocyte lines were stably transfected with the cDNA for human PADPRP in the antisense orientation under an inducible promoter. Induction of this antisense RNA in cultured cells selectively lowers the levels of PADPRP mRNA, protein, and enzyme activity. Induction of antisense RNA also led to a reduction in the levels of PADPRP in individual cell nuclei, as well as the loss of the ability of cells to synthesize and modify proteins by poly(ADP-ribose) polymer in response to DNA damage. When keratinocyte clones containing the antisense construct or empty vector alone were grafted onto nude mice, they formed histologically normal human skin. The PADPRP antisense construct was also inducible in vivo by the topical application of dexamethasone to the reconstituted epidermis. In addition, poly(ADP-ribose) polymer could be induced and detected in vivo following the topical application of a DNA alkylating agent to the grafted transfected skin layers. Accordingly, a model system has been developed in which the levels of PADPRP can be selectively manipulated in human keratinocytes in cell culture, and potentially in reconstituted epidermis as well. This system will be a useful tool to study the role of PADPRP and DNA repair in general in essential biologic processes in the epidermis.

Alkylating Agents↗
PubMed1994

Functional expression of human poly(ADP-ribose) polymerase in Schizosaccharomyces pombe results in mitotic delay at G1, increased mutation rate, and sensitization to radiation.

The activity of poly(ADP-ribose) polymerase (PADPRP), a chromatin-associated enzyme present in most eukaryotic cells, is stimulated by DNA strand breaks, suggesting a role for the enzyme in the cellular response to DNA damage. However, the primary function of PADPRP remains unknown. We have selected Schizosaccharomyces pombe as a simple eukaryotic system in which to study PADPRP function because this fission yeast shares with mammalian cells important cellular features possibly associated with poly-(ADP-ribos)ylation pathways. We investigated the existence of an endogenous yeast PADPRP by DNA and RNA hybridization to mammalian probes under low-stringency conditions and by PADPRP activity assays. Our data indicate that fission yeasts are naturally devoid of PADPRP. We therefore isolated S. pombe strains expressing PADPRP by transformation with a human full-length PADPRP cDNA under the control of the SV40 early promoter. The human PADPRP construct was transcribed and translated in S. pombe, generating a major transcript of the same size (3.7 kb) as that detected in mammalian cells and a 113-kDa polypeptide, identical in size to the native human PADPRP protein. Yeast recombinant PADPRP was enzymatically active and was recognized by antibodies to human PADPRP. S. pombe cells expressing PADPRP (SPT strains) showed a stable phenotype that was characterized by: (i) cell cycle retardation as a result of a specific delay at the G1 phase, (ii) decreased cell viability in stationary cultures, (iii) enhanced rates of spontaneous and radiation-induced ade6-ade7 mutations, and (iv) increased sensitivity to radiation. SPT strains may prove efficient tools with which to investigate PADPRP functions in eukaryotic cells.

Dose-Response Relationship, Radiation↗
PubMed1994

Poly(ADP-ribose) polymerase gene on chromosome 1q: early role in differentiation linked replication; gene on human chromosome 13q: marker of carcinogenesis.

We have focused in part on utilizing molecular aspects of the PADPRP gene sequences located on chromosome 1q as well as on 13 to help clarify the biological role of these genes. With respect to chromosome 1q, stably transfected cell lines have been established in-both HeLa as well as 3T3-L1 preadipocyte cells, which express a reverse complementary transcription product in vivo to the exons coded for on the chromosome 1 locus. Also, in the case of the PADPRP-like gene on chromosome 13q, we have fine mapped this sequence and have observed in a linkage of deletion of this region with predisposition to various forms of cancer, especially in the African-American population.

3T3 Cells↗
PubMed1993

The Malmö biomarker programme.

Evaluation of pro-oxidant/antioxidant dietary factors in relation to individual susceptibility to cancer and cardiovascular disease is justified based on a review of the literature. This working hypothesis is amendable to further scientific validation from biomarkers with end-point sensitivity to oxygen radicals. So far, the biomarker programme developed around this theme may be divided into three distinct classes: (1) Markers of genotoxic exposure estimate DNA damage either directly as a biologically effective dose, or indirectly by estimating aberrant cellular functions that lead to accumulation of DNA damage. The examples included are ADP-ribosylation in mononuclear leucocytes (R. Pero, A. Olsson), oxidative DNA damage (K. Frenkel), gene expression in lymphocytes (S. Garte, G. Cosma), serum alpha macroglobulin (W. Troll) and oxidized DNA damage and repair (N. Christie). (2) Markers of genetic predisposition have been shown to have genetic inheritance patterns that relate to individual susceptibility to cancer or cardiovascular disease. The examples included are glutathione transferase mu phenotyping (R. Pero, J. Seidegård) and poly (ADP-ribose) polymerase pseudogene polymorphism (M. Smulson). (3) Markers of dietary status have been validated to estimate the amount of a particular nutrient or xenobiotic in the diet that has been taken up and metabolized or distributed to body fluids or tissues. The example included here is niacin nutriture (E. Jacobson, M. Jacobson). This biomarker is presented in Section 5 (pp. 59-62) of the Malmö Diet and Cancer Programme Minisymposium reported in this issue of the journal.

Animals↗
PubMed1992

Differences in the poly(ADP-ribosyl)ation patterns of ICP4, the herpes simplex virus major regulatory protein, in infected cells and in isolated nuclei.

Infected-cell protein 4 (ICP4), the major regulatory protein in herpes simplex viruses 1 and 2, was previously reported to accept 32P from [32P]NAD in isolated nuclei. This modification was attributed to poly(ADP-ribosyl)ation (C. M. Preston and E. L. Notarianni, Virology 131:492-501, 1983). We determined that an antibody specific for poly(ADP-ribose) reacts with ICP4 extracted from infected cells, electrophoretically separated in denaturing gels, and electrically transferred to nitrocellulose. Our results indicate that all forms of ICP4 observed in one-dimensional gel electrophoresis are poly(ADP-ribosyl)ated. Poly(ADP-ribose) on ICP4 extracted from infected cells was resistant to cleavage by purified poly(ADP-ribose) glycohydrolase unless ICP4 was in a denatured state. Poly(ADP-ribose) added to ICP4 in isolated nuclei was sensitive to this enzyme. This result indicates that the two processes are distinct and may involve different sites on the ICP4 molecule.

Adenosine Monophosphate↗
PubMed1990

Enhanced poly(adenosine diphosphate ribose) polymerase activity and gene expression in Ewing's sarcoma cells.

Ewing's sarcoma (ES) is a highly malignant childhood bone tumor and is considered curable by moderate doses of radiotherapy. The addition of chemical inhibitors of the activity of the nuclear enzyme poly(adenosine diphosphate ribose) [poly(ADPR)] polymerase to ES cells in culture results in increased cell killing, a phenomenon called "inhibitor sensitization." Since poly(ADPR) polymerase is thought to be associated with DNA repair, it has been suggested that ES cells and other inhibitor-sensitized cells may have a reduced capacity for polymer synthesis resulting in deficient postirradiation recovery. We present here the unexpected observation that in comparison to other cell lines tested, ES cells exhibit a high enzyme activity, higher constitutive levels of the protein, and elevated levels of its mRNA transcript for poly(ADPR) polymerase. No gross amplifications or rearrangements of the gene were observed; however, regulation of poly(ADPR) polymerase in these tumor cells takes place at the level of the gene transcript.

Blotting, Northern↗
PubMed1990

Cell cycle regulation of an exogenous human poly(ADP-ribose) polymerase cDNA introduced into murine cells.

We have evaluated the regulation of expression of the poly(ADP-ribose) polymerase gene during cell growth and replication. In a synchronized population of HeLa cells or in serum-stimulated WI-38 cells, steady-state levels of the polymerase mRNA were highest at late S and S-G2 phases and negligible in early S phase. Transcription did not solely account for the significant increase in the mRNA levels observed in late S phase by Northern analysis. The stability of the mRNA was dependent upon the percent proliferating cells in the culture. Accordingly, polymerase mRNA from cells in early exponential phase was significantly more stable than from cells in stationary phase of asynchronous growth. To clarify these observations, we utilized a novel heterologous expression system that involved murine 3T3 cells transfected with a human poly(ADP-ribose) polymerase cDNA under the control of a non-cell cycle-specific promoter. Cells were synchronized, and a comparison was made of the endogenous (murine) and exogenous (human) polymerase mRNA levels. Both the endogenous and the exogenous mRNA were specifically stabilized by the same mechanisms and only during late S phase; therefore, we concluded that mRNA pools for the polymerase are regulated at the post-transcriptional level. The heterologous expression system confirmed that the post-transcriptional regulation system in the mouse cells can recognize and faithfully regulate the human cDNA in response to the murine cell cycle signals. More importantly, the presence of extra copies (human) of the polymerase gene did not provide an increased amount of the total polymerase mRNA or protein and, in fact, the sum of the endogenous and exogenous mRNA in the transfected cells was approximately the same as the level of endogenous transcript in the control cells. This suggested that there might be a limit to the amount of polymerase protein accumulating in the cellular pool and thus levels of poly(ADP-ribose) polymerase may be autoregulated.

Animals↗