Maximal DNA Synthesis Research Articles

Genomics of Human Papillomavirus-induced Cervical Cancer

Human papillomavirus (HPV) induced cervical cancer is a serious health issue among most women from the least developed countries of the world due to scarcity of resources. HPVs have evolved a masterly infectious cycle that grabs the advantage of the self-renewal property of stratified cutaneous and mucosal epithelia. Firstly, the viral genome replicates episomally at a low copy number in the epithelial basal layers cells, with minimum viral transcription and translation. After that, when the infected cells are transmitted through the differentiation process maximum viral DNA synthesis and gene expression occur. The two major oncoproteins - E6 and E7, are responsible for inactivating the important tumor suppressor proteins, retinoblastoma (pRb) and p53. Due to the inactivation of these proteins, disruption occurs in the DNA replication, DNA repair mechanisms, oxidative induced damage (8-oxoguanine), aneuploidy, and apoptosis, leading to tumorigenesis. Hence, manipulation of E6 and E7 genes shows a successful result in the treatment of cervical cancer. The diagnosis tests include liquid-based preparations to improve the standard of the Pap smear; computer-based screening methods to improve Pap smear interpretation; and HPV testing methods that may be beneficial in triaging patients with untypical squamous cells of unknown significance or low grade squamous intraepithelial lesions (SILs). The continued studies of the molecular biology of HPVs are necessary to develop advanced screening techniques for testing and prophylactic vaccines for the elimination of HPV infection, also better therapeutic vaccines for the treatment of various HPVs infections.

Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates

SummaryThe integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo.

Folate-Dependent Purine Nucleotide Biosynthesis in Humans.

Purine nucleotide biosynthesis de novo (PNB) requires 2 folate-dependent transformylases-5'-phosphoribosyl-glycinamide (GAR) and 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) transformylases-to introduce carbon 8 (C8) and carbon 2 (C2) into the purine ring. Both transformylases utilize 10-formyltetrahydrofolate (10-formyl-H4folate), where the formyl-carbon sources include ring-2-C of histidine, 3-C of serine, 2-C of glycine, and formate. Our findings in human studies indicate that glycine provides the carbon for GAR transformylase (exclusively C8), whereas histidine and formate are the predominant carbon sources for AICAR transformylase (C2). Contrary to the previous notion, these carbon sources may not supply a general 10-formyl-H4folate pool, which was believed to equally provide carbons to C8 and C2. To explain these phenomena, we postulate that GAR transformylase is in a complex with the trifunctional folate-metabolizing enzyme (TFM) and serine hydroxymethyltransferase to channel carbons of glycine and serine to C8. There is no evidence for channeling carbons of histidine and formate to AICAR transformylase (C2). GAR transformylase may require the TFM to furnish 10-formyl-H4folate immediately after its production from serine to protect its oxidation to 10-formyldihydrofolate (10-formyl-H2folate), whereas AICAR transformylase can utilize both 10-formyl-H2folate and 10-formyl-H4folate. Human liver may supply AICAR to AICAR transformylase in erythrocytes/erythroblasts. Incorporation of ring-2-C of histidine and formate into C2 of urinary uric acid presented a circadian rhythm with a peak in the morning, which corresponds to the maximum DNA synthesis in the bone marrow, and it may be useful in the timing of the administration of drugs that block PNB for the treatment of cancer and autoimmune disease.

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.

Phenotypes and circadian rhythm in utilization of formate in purine nucleotide biosynthesis de novo in adult humans

Aims Folate coenzymes and dependent enzymes introduce one carbon units at positions 2 (C 2) and 8 (C 8) of the purine ring during de novo biosynthesis. Formate is one source of one-carbon units. Although much is known about lower organisms, little data exists describing formate utilization for purine biosynthesis in humans. Main methods Mass-spectrometric analysis of urinary uric acid, the final purine catabolite, following 1.0 g oral doses of sodium [ 13C] formate was performed and detected 13C enrichment at C 2 and C 8 separately. Key findings Three phenotypes were suggested. One incorporates 13C 0.72 to 2.0% into C 2 versus only 0 to 0.07% into C 8. Another incorporates only 0 to 0.05% 13C into C 2 or C 8. A third phenotype incorporates 13C into C 8 (0.15%) but C 2 incorporation (0.44%) is still greater. In subjects who incorporated 13C formate into C 2, peak enrichment occurred in voids from 8–12 h (24 h clock) suggesting a circadian rhythm. Significance Evidence that mammalian liver introduces C 8 and that C 2 is introduced in a non-hepatic site would explain our results. Our data are not similar to those in non-mammalian organisms or cells in culture and are not consistent with the hypothesis that formate from folate-dependent metabolism in mitochondria is a major one carbon source for purine biosynthesis. Timing of peak 13C enrichment at C 2 corresponds to maximal DNA synthesis in human bone marrow. Phenotypes may explain the efficacy (or lack of) of certain anticancer and immunosuppressive drugs.

Abstract 400: Establishment and characterization of a novel IL6-dependent HLA-A2+ human myeloma cell line LNT1

Abstract The novel multiple myeloma (MM) cell line LNT1 was established from peripheral blood of a 66-year-old female patient with recurrent plasma cell leukemia of IgG/kappa type. Peripheral blood mononuclear cells from the heparinized peripheral blood separated by Ficoll-Hypaque density gradient centrifugation were cultured in RPMI 1640 medium/20% FBS, 100 U/ml penicillin and 50 μg/ml streptomycin, without additional growth factors. Cells were cultured by replacing ½ of the volume of the culture with fresh medium every 3-4 days for 3 months. In the 4th month, a slow yet sustained growth of cultured cells was noted, and designated LNT1. These cells grow as single cell suspension; IL-6-induced proliferation in a dose-dependent manner with 2 ng/ml of IL-6 inducing maximal DNA synthesis (by [3H]thymidine uptake) and survival (by MTT). In contrast, IGF-1 had no effect. Subsequently, 2 ng/ml of IL-6 was added to cultures and growth now sustained at 7 months. LNT1 was negative for Epstein-Barr virus, as evidenced by PCR using primers specific for EBNA-1. May-Grunwald-Giemsa staining showed that LNT1 cells have a high nucleocytoplasmic ratio with excentrically located nuclei, prominent nucleoli, and a deeply basophilic and vacuolated cytoplasm. Some cells were bi- or multi-nucleated. Surface antigen analysis showed that LNT1 cells express CD138, CD38, HM1.24, CS1, and HLA-A2, but did not express CD19, CD20, CD3, CD16, and CD14. Chromosome analysis at 7 months revealed the karyotype: 43, X, -X, del(1)(p13p22), psu dic(6;1)(q13;p11), del(12)(p11.2), −13, add(19)(q13.4), −22[2]/43, idem, del(2)(q11.2q2?3), der(6)ins(6;?)(q21;?)[7]. DNA fingerprinting was performed by simultaneously amplifying eight short tandem repeat (STR) loci and the amelogenin gene in a multiplex PCR reaction using specific primers to confirm the authentication of cell line. The established LNT1 cell line and the primary tumor showed identical band patterns of these 9 markers and the same immunoglobulin H rearrangement. An unique and identical VH3/IgG fragment was amplified using the appropriate VH family-specific framework region primer in conjunction with CH isotype specific (IgG) primers in DNA samples of both LNT1 cell line and the original tumor. Moreover, identical DNA sequence of VH3/IgG PCR from cell line and primary tumor further confirms clonality. LNT1 cell line also bears a N-RAS G12D mutation, as in the original tumor. Finally, LNT1 cell line is responsive to conventional (Dex, Malphalan) and novel or emerging (bortezomib, lenalidomide, perifosine, AS703026) anti-MM therapies. Therefore, the novel LNT1 cell line will provide a useful model system to study genetics and biology of MM, as well as to evaluate novel therapeutic strategies. Since LNT1 cells highly express HLA-A2, this line also serves a useful model to validate novel tumor-associated antigens for immunotherapy in MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 400.

CYTOGENETIC STUDIES OF X-RAY AND INGESTED P32 INDUCED SEX-LINKED RECESSIVE LETHALS IN DROSOPHILA MELANOGASTER

Male flies were either irradiated with 1000 r or fed a sugar solution containing Pa/sup 32/. The latter flies attained an average activity of 75 C. The males were immediately mated with unirradiated females according to the brood pattern technique. Chromosomal aberrations (deletions, translocations, and inversions) were recovered following treatment with either mutagen. The distribution of point mutation lethals induced on the X chromosome by P/sup 32/ was identical to the one found after x-ray treatment. The highest frequency of sex-linked recessive lethals in the P/sup 32/ series was found on days 7 and 8, as compared with day 5 for the x-ray series. The sterile period in the x-ray series following the exhaustive mating system used may be attributed partially to dominant lethals, lack of functional sperm, and to meiotic or mitotic inhibition due to irradiation. P/sup 32/ incorporated into the DNA of mature sperm was recovered 7 to 8 days after a single meal, under conditions of excessive mating, which coincides with maximum genetic effect. Thus the results favor the concept that the period of maximum DNA synthesis corresponds to the period of maximum lethal induction and/or of chromosome breakage. (H.H.D.)

In vivo replication kinetics and transcription patterns of the nucleopolyhedrovirus (NeabNPV) of the balsam fir sawfly, Neodiprion abietis

DNA replication and transcription of NeabNPV, the nucleopolyhedrovirus (NPV) of the balsam fir sawfly, Neodiprion abietis (Hymenoptera: Diprionidae), in host larvae were investigated. NPV DNA replication kinetics and gene-expression patterns have been resolved only in lepidopteran cell-culture systems and in limited in vivo experiments with lepidopteran larvae. Furthermore, there are significant differences in pathologies caused by lepidopteran NPVs, which replicate in many tissues, and hymenopteran NPVs, known to replicate in midgut epithelium only. Despite the differences in host specificity and pathology, NeabNPV DNA replication kinetics were similar to those reported for lepidopteran NPVs. Maximal NeabNPV DNA synthesis was observed between 4 and 24 h post-inoculation (p.i.) but, in contrast to lepidopteran NPVs, synthesis continued at a lower rate up to 72 h p.i. Selected NeabNPV genes exhibited a cascade pattern of transcription similar to that of lepidopteran NPVs. RT-PCR products of the NeabNPV lef-1, lef-2 and dnapol transcripts were observed as early as 2 h p.i., whilst lef-8 and lef-9, encoding putative viral RNA polymerase subunits, were detected at 1 and 6 h p.i., respectively. Two structural late transcripts (gp41 and p74) were observed from 6 h p.i. The very late factor 1 (vlf-1) transcript, a transactivator of very late genes, was observed from 12 h p.i., but the very late transcript polh, encoding the major occlusion protein, polyhedrin, was observed from 24 h p.i. This study provides the first insight into DNA replication and gene expression of a non-lepidopteran baculovirus.

Aging does not reduce the hepatocyte proliferative response of mice to the primary mitogen TCPOBOP.

It has been shown that the magnitude of DNA synthesis and the time at which maximal DNA synthesis occurs after two-thirds partial hepatectomy (PH) is greatly reduced in the liver of aged rodents compared to young animals. This reduction could represent an intrinsic defect in proliferation or a more specialized change in the response to PH. We therefore evaluated the proliferative capacity of hepatocytes in aged animals, following treatment with primary liver mitogens. We show that treatment of 12-month-old CD-1 mice with the hepatomitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) caused an increase in hepatocyte proliferation similar to that seen in young (8-week-old) mice. The labeling index was 82% in the livers of aged mice versus 76% in young animals. Histological observation demonstrated that the number of hepatocytes entering mitoses was similar in both groups; the mitotic indices were 2.5 per thousand and 2.7 per thousand, respectively. Additional experiments showed that the timing of DNA synthesis and M phase were nearly identical in both aged and young mice. Stimulation of hepatocyte DNA synthesis was associated with increased expression of several cell cycle-associated proteins (cyclin D1, cyclin A, cyclin B1, E2F, pRb, and p107); all were comparable in aged mice and young mice. TCPOBOP treatment also increased expression of the Forkhead Box transcription factor m1b (Foxm1b) to a similar degree in both groups. In conclusion, hepatocytes retain their proliferative capacity in old age despite impaired liver regeneration. These findings suggest that therapeutic use of mitogens would alleviate the reduction in hepatocyte proliferation observed in the elderly.

Zonal differences in effects of HGF/SF and EGF on DNA synthesis in hepatocytes at different times post-hepatectomy

This study was done for investigation of the DNA synthesis induced by HGF/SF and EGF in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) isolated at different times post-hepatectomy. Preparation, culture, and DNA synthesis of PPH and PVH were done as previously described. In both PPH and PVH isolated at 6 and 12 h post-hepatectomy, the maximum DNA synthesis induced by both HGF/SF and EGF was observed from 24 to 48 h post-hepatectomy. In contrast, in both types of hepatocytes isolated at 36, 72 and 96 h, 1 and 2 weeks, and 1 month post-hepatectomy, maximum DNA synthesis was observed from 48 to 72 h. However, maximum DNA synthesis induced by HGF/SF and EGF was at its lowest in PPH isolated at 12 h and PVH isolated at 6 h post-hepatectomy. Maximum DNA synthesis returned to the control level in PPH and PVH isolated at 72 and 96 h post-hepatectomy, respectively. Specific binding studies using [ 125 I ] HGF/SF and [ 125 I ] EGF supported the above data. From these data, it was demonstrated that first, the PPH and PVH cell cycles have the same post-hepatectomy, but are dependent on the preparation time. Secondly, differences in DNA synthesis in PPH and PVH post-hepatectomy suggest that liver regeneration may occur unevenly.

Tumor Necrosis Factor-α Acts as a Complete Mitogen for Primary Rat Hepatocytes

The cytokine tumor necrosis factor (TNF)-alpha has previously been shown to prime hepatocytes to a state of replicative competence, but has not been shown to act as a complete mitogen for these cells. In the present study we have altered our previously described long-term dimethyl sulfoxide culture system to exclude all known hepatocyte mitogens from the culture media and enable us to directly examine the effects of TNF-alpha on primary rat hepatocytes. We have shown that cells maintained under these culture conditions retain the biochemical and morphological features of well-differentiated hepatocytes. Treatment with TNF-alpha induced DNA synthesis relative to control, to a level not significantly different from that induced by the known hepatocyte mitogen, epidermal growth factor (EGF). Maximal DNA synthesis was induced by treatment with 250 U/ml TNF-alpha for 24 hours. Mitotic figures were observed in cultures treated with TNF-alpha or EGF but not in untreated controls. Treatment of cultures with TNF-alpha, but not EGF, induced activation of both nuclear factor-kappaB p50 homodimers and p50/p65 heterodimers. DNA synthesis induced by TNF-alpha was inhibited by treatment with transforming growth factor-beta. Based on the results of our studies, we conclude that TNF-alpha acts as a complete mitogen for rat hepatocytes.

Changes in mitochondrial adenine nucleotides and in permeability transition in two models of rat liver regeneration

Although enhanced phosphorylative activity can be a requisite for later DNA synthesis during liver regeneration (LR), mitochondrial generation of reactive oxygen species could lead to altered mitochondrial membrane permeability during the prereplicative phase of LR. Therefore, the role of mitochondrial permeability transition (MPT) was evaluated during rat LR, induced by either partial hepatectomy (PH) or after CCl 4 administration. Parameters indicative of mitochondrial function and membrane potentials, those of oxidative stress, and in vivo changes of the intramitochondrial pool of adenine nucleotides were determined. Twelve hours after PH, mitochondrial oxidative and phosphorylative activities and adenosine diphosphate (ADP) content were increased, reaching a maximal peak at 24 hours after surgery (maximal DNA synthesis). Parameters suggestive of oxidant stress were enhanced, but mitochondrial volume and membrane electrical potential remained unaltered. Interestingly, moderate mitochondrial swelling and depolarization were found at later post-PH times (72 hours). In CCl 4-treated animals, it was found that an active liver cell necrosis delayed mitotic activity and mitochondrial uncoupled respiration. Starting 12 hours after CCl 4 intoxication, a drastic increase of inorganic phosphate occurred within swollen and strongly depolarized mitochondria, suggesting changes in the MPT. Despite expression of messenger RNA (mRNA) for mitochondrial transcription, factor A showed a similar time course in both experimental models. The so-called augmenter liver regeneration was found significantly elevated only in PH rats. In conclusion, onset of MPT could be associated with cell necrosis and inflammation after CCl 4 treatment, whereas this mitochondrial event could constitute a putative effector mechanism, through which growth or inflammatory factors inhibiting cell proliferation could initiate LR termination. (H epatology 2003;37:842-851.)

Interleukin-6/Glycoprotein 130-dependent Pathways Are Protective during Liver Regeneration

After tissue loss the liver has the unique capacity to restore its mass by hepatocyte proliferation. Interleukin-6 (IL6)-deficient mice show a lack in DNA synthesis after partial hepatectomy (PH). To define better the role of IL6 and its family members for liver regeneration after PH, we used conditional knockout mice for glycoprotein 130 (gp130), the common signal transducer of all IL6 family members. We show that gp130-dependent pathways control Stat3 activation after PH. By using gene array analysis, we demonstrate that c-jun, NF-kappa B, c-myc, and tumor necrosis factor receptor expression is gp130-dependent. However, in gp130-deleted mice only minor effects on cell cycle and on the maximum of DNA synthesis after PH were found compared with controls. As in conditional gp130 animals, the acute phase response was completely abolished, we considered that other means are essential to define the role of gp130-dependent pathways for liver regeneration. LPS stimulation in gp130-deleted and also IL6 -/- animals after PH leads to a significant reduction in survival and DNA synthesis, which was associated with decreased Bcl-xL expression and higher apoptosis in the liver. These results indicate that the phenotype concerning the reduction in DNA synthesis might be linked to the degree of infection after PH. Thus our results suggest that the role of gp130-dependent signaling is not a direct influence on cell cycle progression after partial hepatectomy but is to activate protective pathways important to enable hepatocyte proliferation.

Differences in iron requirements by concanavalin A-treated and anti-CD3-treated murine splenic lymphocytes.

Fe availability is critical for optimal lymphocyte proliferation; however, the minimum required levels are unknown. Such information is valuable when assessing in vitro immune responses in Fe-deficient subjects, because serum (Fe) added to the culture medium may replete lymphocytes. To address this issue, splenic lymphocytes obtained from seventeen 3-month-old C57BL/6 mice were incubated without and with 1 mg/l concanavalin A or 50 microg/l anti-CD3 antibody in media that contained between 0.113 and 9.74 micromol Fe/l. Fe was provided by either fetal calf serum (FCS, 0-100 ml/l), newborn calf serum (NBCS, 0-100 ml/l), or NBCS (10 ml/l) plus ferric ammonium citrate. As expected, the rate of DNA synthesis increased with Fe levels (P<0.01). Maximum DNA synthesis was obtained with 2.26 micromol Fe/l (50 ml FCS/l) for concanavalin A and 0.895 micromol/l (20 ml FCS/l) for anti-CD3-treated cells. In serum-free media (0.113 micromol Fe/l), the proliferative responses to concanavalin A were below the background, while they rose 5.5-fold in anti-CD3-treated cells (P<0.05). In apotransferrin-supplemented media (0.13 micromol Fe/l), the proliferative responses to concanavalin A and anti-CD3 antibody were 18.6 and 71 %, respectively, of that obtained with 4.66 micromol Fe/l (100 ml FCS/l). Interleukin 2 secretion also followed the same trend as lymphocyte proliferation. Since differences between both mitogens persisted after FCS was substituted with NBCS, we can rule out an effect on ribonucleotide reductase activity, or by other serum growth factors. We speculate an Fe effect at an early step of T-cell activation. Data suggest that the minimum Fe concentration required for lymphocyte proliferation varies with the mitogen.

Differences in iron requirements by concanavalin A-treated and anti-CD3-treated murine splenic lymphocytes

Fe availability is critical for optimal lymphocyte proliferation; however, the minimum required levels are unknown. Such information is valuable when assessingin vitroimmune responses in Fe-deficient subjects, because serum (Fe) added to the culture medium may replete lymphocytes. To address this issue, splenic lymphocytes obtained from seventeen 3-month-old C57BL/6 mice were incubated without and with 1 mg/l concanavalin A or 50 μg/l anti-CD3 antibody in media that contained between 0·113 and 9·74 μmol Fe/l. Fe was provided by either fetal calf serum (FCS, 0–100 ml/l), newborn calf serum (NBCS, 0–100 ml/l), or NBCS (10 ml/l) plus ferric ammonium citrate. As expected, the rate of DNA synthesis increased with Fe levels (P&lt;0·01). Maximum DNA synthesis was obtained with 2·26 μmol Fe/l (50 ml FCS/l) for concanavalin A and 0·895 μmol/l (20 ml FCS/l) for anti-CD3-treated cells. In serum-free media (0·113 μmol Fe/l), the proliferative responses to concanavalin A were below the background, while they rose 5·5-fold in anti-CD3-treated cells (P&lt;0·05). In apotransferrin-supplemented media (0·13 μmol Fe/l), the proliferative responses to concanavalin A and anti-CD3 antibody were 18·6 and 71 %, respectively, of that obtained with 4·66 μmol Fe/l (100 ml FCS/l). Interleukin 2 secretion also followed the same trend as lymphocyte proliferation. Since differences between both mitogens persisted after FCS was substituted with NBCS, we can rule out an effect on ribonucleotide reductase activity, or by other serum growth factors. We speculate an Fe effect at an early step of T-cell activation. Data suggest that the minimum Fe concentration required for lymphocyte proliferation varies with the mitogen.

Nuclear Translocation of Mouse Polycomb M33 Protein in Regenerating Liver

Immunoblots probed with an antibody to M33 protein, a homolog of Drosophila Polycomb, revealed that most M33 in adult mouse liver had a higher electrophoretic mobility than that in F9 embryonal carcinoma cells. High-mobility 60-kDa M33 localized in the cytoplasmic fraction of liver homogenates, and two less abundant 66- and 70-kDa species were detected in the nuclear fraction. Immunocytochemistry of freeze-substituted tissues showed a punctate pattern of immunofluorescence in the cytoplasm of hepatic parenchymal cells. Nuclear M33 isoforms treated with alkaline phosphatase had increased mobilities corresponding to cytoplasmic M33. In partially hepatectomized mice, nuclear M33 isoforms appeared after 48 h, near the time of maximum DNA synthesis as measured by bromodeoxyuridine incorporation. By 60 h, most M33 was in the form of these low-mobility species, and the pattern of immunofluorescence suggested the existence of chromatin-bound and free states of the protein in the nucleus. Thereafter, high-mobility 60-kDa M33 reappeared. The data are consistent with a phosphorylation-associated translocation mechanism that is a cell cycle-dependent.

The DNA-polymerase inhibiting activity of poly(beta-l-malic acid) in nuclear extract during the cell cycle of Physarum polycephalum.

The naturally synchronous plasmodia of myxomycetes synthesize poly(beta-l-malic acid), which carries out cell-specific functions. In Physarum polycephalum, poly(beta-l-malate) [the salt form of poly(beta-l-malic acid)] is highly concentrated in the nuclei, repressing DNA synthetic activity of DNA polymerases by the formation of reversible complexes. To test whether this inhibitory activity is cell-cycle-dependent, purified DNA polymerase alpha of P. polycephalum was added to the nuclear extract and the activity was measured by the incorporation of [3H]thymidine 5'-monophosphate into acid precipitable nick-activated salmon testis DNA. Maximum DNA synthesis by the reporter was measured in S-phase, equivalent to a minimum of inhibitory activity. To test for the activity of endogenous DNA polymerases, DNA synthesis was followed by the highly sensitive photoaffinity labeling technique. Labeling was observed in S-phase in agreement with the minimum of the inhibitory activity. The activity was constant throughout the cell cycle when the inhibition was neutralized by the addition of spermidine hydrochloride. Also, the concentration of poly(beta-l-malate) did not vary with the phase of the cell cycle [Schmidt, A., Windisch, C. & Holler, E. (1996) Nuclear accumulation and homeostasis of the unusual polymer poly(beta-l-malate) in plasmodia of Physarum polycephalum. Eur. J. Cell Biol. 70, 373-380]. To explain the variation in the cell cycle, a periodic competition for poly(beta-l-malate) between DNA polymerases and most likely certain histones was assumed. These effectors are synthesized in S-phase. By competition they displace DNA polymerase from the complex of poly(beta-l-malate). The free polymerases, which are no longer inhibited, engage in DNA synthesis. It is speculated that poly(beta-l-malate) is active in maintaining mitotic synchrony of plasmodia by playing the mediator between the periodic synthesis of certain proteins and the catalytic competence of DNA polymerases.

Cyclin D1 antisense oligonucleotide inhibits cell growth stimulated by epidermal growth factor and induces apoptosis of gastric cancer cells.

The cyclin Dl protein is one of the cell cycle regulators required for cell cycle progression through Gl phase to S phase. The cyclin Dl‐cyclin‐dependent kinase (CDK) system is thought to control the cell cycle through mediating extracellular signals from mitogens, such as epidermal growth factor (EGF). In this study, we attempted to examine the therapeutic effect of cyclin Dl antisense oligonucleotides (AS/D1) on cell proliferation and apoptosis of the gastric cancer cell line MKN‐74, in the presence and absence of EGF‐stimulation. Evaluation of cell survival and DNA synthesis revealed that enhanced cell growth following EGF‐stimulation was completely inhibited by a 24 h pre‐incubation with 100 nM AS/D1. This inhibition was down to 19.3% compared with maximal DNA synthesis after stimulation with 3 nM EGF alone. Western blotting demonstrated that while EGF‐stimulation led to cyclin Dl over‐expression, AS/D1 inhibited cyclin Dl protein expression. We also demonstrated the induction of apoptosis in MKN‐74 cells by AS/D1. In conclusion, EGF‐stimulated MKN‐74 cell proliferation was inhibited by AS/D1, which could overcome EGF‐induced cyclin Dl over‐expression. AS/D1 also affected cell survival by inducing apoptosis through cell cycle arrest following cyclin Dl depletion. Thus, AS/D1 may be a candidate for use as a novel cancer therapy specifically targeted against the over‐expression of cyclin Dl enhanced by EGF in malignant cells

Mesothelial regeneration is not dependent on subserosal cells

It has been proposed that after mesothelial injury, resident cells within the subserosal connective tissue proliferate, differentiate, and migrate to the serosal surface. The aim of this study was to examine the temporal and spatial changes of proliferating cells in a murine model of testicular mesothelial healing and assess the potential of submesothelial cells to reconstitute the damaged mesothelium. Histology and autoradiography were employed to determine the number of cells within the submesothelial connective tissue, as well as the proportion of cells undergoing DNA synthesis on and beneath the injured serosa. Mesothelial cells surrounding the wound demonstrated maximal DNA synthesis 48 h after injury (27.82±5.64% SEM, compared with 0.17±0.16% 3H-TdR labelled cells for resting mesothelium), whereas a significant increase in proliferating submesothelial cells was not seen until day 4 post-injury (7.79±3.31% compared with 0.85±0.64% 3H-TdR labelled cells at day 2). Furthermore, this small number of dividing submesothelial cells must include cells other than the proposed mesothelial precursors, indicating a very low proportion of precursor cells in the submesothelial cell population. As large numbers of mesothelial cells were seen at the wound centre by 3–4 days after injury, it is unlikely that submesothelial cells contributed significantly to the repopulation of the injured mesothelium. It is hypothesized that regenerating mesothelium is more likely to originate from the surrounding uninjured mesothelial cell population. Copyright © 2000 John Wiley & Sons, Ltd.

Mesothelial regeneration is not dependent on subserosal cells.

It has been proposed that after mesothelial injury, resident cells within the subserosal connective tissue proliferate, differentiate, and migrate to the serosal surface. The aim of this study was to examine the temporal and spatial changes of proliferating cells in a murine model of testicular mesothelial healing and assess the potential of submesothelial cells to reconstitute the damaged mesothelium. Histology and autoradiography were employed to determine the number of cells within the submesothelial connective tissue, as well as the proportion of cells undergoing DNA synthesis on and beneath the injured serosa. Mesothelial cells surrounding the wound demonstrated maximal DNA synthesis 48 h after injury (27. 82+/-5.64% SEM, compared with 0.17+/-0.16% (3)H-TdR labelled cells for resting mesothelium), whereas a significant increase in proliferating submesothelial cells was not seen until day 4 post-injury (7.79+/-3.31% compared with 0.85+/-0.64% (3)H-TdR labelled cells at day 2). Furthermore, this small number of dividing submesothelial cells must include cells other than the proposed mesothelial precursors, indicating a very low proportion of precursor cells in the submesothelial cell population. As large numbers of mesothelial cells were seen at the wound centre by 3-4 days after injury, it is unlikely that submesothelial cells contributed significantly to the repopulation of the injured mesothelium. It is hypothesized that regenerating mesothelium is more likely to originate from the surrounding uninjured mesothelial cell population.