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ObjectivesTo investigate how the quality of maternal health services and travel times to health facilities affect birthing service utilisation in Eastern Region, Ghana.DesignThe study is a cross-sectional spatial interaction analysis of birth service utilisation patterns. Routine birth data were spatially linked to quality care, service demand and travel time data.Setting131 Health facilities (public, private and faith-based) in 33 districts in Eastern Region, Ghana.ParticipantsWomen who gave birth in health facilities in the Eastern Region, Ghana in 2017.Outcome measuresThe count of women giving birth, the quality of birthing care services and the geographic coverage of birthing care services.ResultsAs travel time from women’s place of residence to the health facility increased up to two2 hours, the utilisation rate markedly decreased. Higher quality of maternal health services haves a larger, positive effect on utilisation rates than service proximity. The quality of maternal health services was higher in hospitals than in primary care facilities. Most women (88.6%) travelling via mechanised transport were within two2 hours of any birthing service. The majority (56.2%) of women were beyond the two2 -hour threshold of critical comprehensive emergency obstetric and newborn care (CEmONC) services. Few CEmONC services were in urban centres, disadvantaging rural populations.ConclusionsTo increase birthing service utilisation in Ghana, higher quality health facilities should be located closer to women, particularly in rural areas. Beyond Ghana, routinely collected birth records could be used to understand the interaction of service proximity and quality.

Following the recent expiry of the United Nations’ 2015 Millennium Development Goals (MDGs), new international development agenda covering 2030 water, sanitation and hygiene (WASH) targets have been proposed, which imply new demands on data sources for monitoring relevant progress. This study evaluates drinking-water and sanitation classification systems from national census questionnaire content, based upon the most recent international policy changes, to examine national population census’s ability to capture drinking-water and sanitation availability, safety, accessibility, and sustainability. In total, 247 censuses from 83 low income and lower-middle income countries were assessed using a scoring system, intended to assess harmonised water supply and sanitation classification systems for each census relative to the typology needed to monitor the proposed post-2015 indicators of WASH targets. The results signal a lack of international harmonisation and standardisation in census categorisation systems, especially concerning safety, accessibility, and sustainability of services in current census content. This suggests further refinements and harmonisation of future census content may be necessary to reflect ambitions for post-2015 monitoring.

Murine Pre-13 lymphocytes with experimentally activated MycER show both chromosomal and extrachromosomal gene amplification. In this report, we have elucidated the size, structure, functional components of c-Myc-induced extrachromosomal elements (EEs). Scanning electron microscopy revealed that EEs isolated from MycER-activated Pre-B+ cells are an average of 10 times larger than EEs isolated from non-MycER-activated control Pre-B- cells. We demonstrate that these large c-Myc-induced EEs are associated with histone proteins, whereas EEs of non-MycER-activated Pre B- cells are not. Immunohistochemistry and Western blot analyses using pan -histone-specific, histone H3 phosphorylation-specific, histone H4 acetylation-specific antibodies indicate that a significant proportion of EEs analyzed from MycER-activated cells harbors transcriptionally competent and/or active chromatin. Moreover, these large, c-Myc-induced EEs carry genes. Whereas the total genetic make-up of these c-Myc-induced EEs is unknown, we found that 30.2% of them contain the dihydrofolate reductase (DHFR) gene, whereas cyclin C (CCNC) was absent. In addition, 50% of these c-Myc-activated Pre-B+ EEs incorporated bromodeoxyuridine (BrdU), identifying them as genetic structures that self-propagate. In contrast, EEs isolated from non-Myc-activated cells neither carry the DHFR gene nor incorporate BrdU, suggesting that c-Myc deregulation generates a new class of EEs.

The c-Myc oncoprotein is highly expressed in malignant cells of many cell types, but the mechanism by which it contributes to the transformation process is not fully understood. Here, we show for the first time that constitutive or activated overexpression of the c-myc gene in cultured mouse B lymphocytes is followed by chromosomal and extrachromosomal amplification as well as rearrangement of the ribonucleotide reductase R2 gene locus. Electron micrographs and fluorescent in situ hybridization (FISH) demonstrate the c-Myc-dependent generation of extrachromosomal elements, some of which contain R2 sequences. However, unlike other genes that have been shown to be targets of c-Myc-dependent genomic instability, amplification of the R2 gene is not associated with alterations in R2 mRNA or protein expression. These data suggest that c-Myc-dependent genomic instability involves a greater number of genes than previously anticipated, but not all of the genes that are amplified in this system are transcriptionally upregulated.

When the Ras mitogen-activated protein kinase (MAPK) signaling pathway of quiescent cells is stimulated with growth factors or phorbol esters, the early response genes c-fos and c-myc are rapidly induced, and concurrently there is a rapid phosphorylation of histone H3. Using an antibody specific for phosphorylated Ser-10 of H3, we show that Ser-10 of H3 is phosphorylated, and we provide direct evidence that phosphorylated H3 is associated with c-fos and c-myc genes in stimulated cells. H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation. Previously we reported that persistent stimulation of the Ras-MAPK signaling pathway in oncogene-transformed cells resulted in increased amounts of phosphorylated histone H1. Here we show that phosphorylated H3 is elevated in the oncogene-transformed mouse fibroblasts. Further we show that induction of ras expression results in a rapid increase in H3 phosphorylation. H3 phosphatase, identified as PP1, activities in ras-transformed and parental fibroblast cells were similar, suggesting that elevated H3 kinase activity was responsible for the increased level of phosphorylated H3 in the oncogene-transformed cells. Elevated levels of phosphorylated H1 and H3 may be responsible for the less condensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells.

Ribonucleotide reductase is a rate-limiting enzyme in DNA synthesis and is composed of two different proteins, R1 and R2. The R2 protein appears to be rate-limiting for enzyme activity in proliferating cells, and it is phosphorylated by p34cdc2 and CDK2, mediators of cell cycle transition events. A sequence in the R2 protein at serine-20 matches a consensus sequence for p34cdc2 and CDK2 kinases. We tested the hypothesis that the serine-20 residue was the major p34cdc2 kinase site of phosphorylation. Three peptides were synthesized (from Asp-13 to Ala-28) that contained either the wild type amino acid sequence (Asp-Gln-Gln-Gln-Leu-Gln-Leu-Ser-Pro-Leu-Lys-Arg-Leu-Thr-Leu-Ala, serine peptide) or a mutation, in which the serine residue was replaced with an alanine residue (alanine peptide) or a threonine residue (threonine peptide). Only the serine peptide and threonine peptide were phosphorylated by p34cdc2 kinase. In two-dimensional phosphopeptide mapping experiments of serine peptide and Asp-N endoproteinase digested R2 protein, peptide co-migration patterns suggested that the synthetic phosphopeptide containing serine-20 was identical to the major Asp-N digested R2 phosphopeptide. To further test the hypothesis that serine-20 is the primary phosphorylated residue on R2 protein, three recombinant R2 proteins (R2-Thr, R2-Asp and R2-Ala) were generated by site-directed mutagenesis, in which the serine-20 residue was replaced with threonine, aspartic acid or alanine residues. Wild type R2 and threonine-substituted R2 proteins (R2-Thr) were phosphorylated by p34cdc2 kinase, whereas under the same experimental conditions, R2-Asp and R2-Ala phosphorylation was not detected. Furthermore, the phosphorylated amino acid residue in the R2-Thr protein was determined to be phosphothreonine. Therefore, by replacing a serine-20 residue with a threonine, the phosphorylated amino acid in R2 protein was changed to a phosphothreonine. In total, these results firmly establish that a major p34cdc2 phosphorylation site on the ribonucleotide reductase R2 protein occurs near the N-terminal end at serine-20, which is found within the sequence Ser-Pro-Leu-Lys-Arg-Leu. Comparison of ribonucleotide reductase activities between wild type and mutated forms of the R2 proteins suggested that mutation at serine-20 did not significantly affect enzyme activity.

An altered nuclear morphology has been previously noted in association with Ras activation, but little is known about the structural basis, functional significance, signaling pathway, or reproducibility of any such change. We first tested the reproducibility of Ras-associated nuclear change in a series of rodent fibroblast cell lines. After independently developing criteria for recognizing Ras-associated nuclear change in a Papanicolaou stained test cell line with an inducible H(T24)-Ras oncogene, two cytopathologists blindly and independently assessed 17 other cell lines. If the cell lines showed Ras-associated nuclear change, a rank order of increasing nuclear change was independently scored. Ras-associated nuclear changes were identified in v-Fes, v-Src, v-Mos, v-Raf, and five of five H(T24)-Ras transfectants consisting of a change from a flattened, occasionally undulating nuclear shape to a more rigid spherical shape and a change from a finely textured to a coarse heterochromatic appearance. Absent or minimal changes were scored in six control cell lines. The two cytopathologists' independent morphologic rank orders were similar (P .11). The rank order correlated closely with metastatic potential (P < .0014 and P < .0003) but not with histone H1 composition or global nuclease sensitivity. Based on published studies of five of the cell lines, there may be a correlation between increases in certain nuclear matrix proteins and the Ras-associated nuclear change.

Fully thioated antisense molecules are often cytotoxic and non-specitic in action. GPI2A is thioated at 7 base positions. GPI2A posses sequence-specific activity against HIV-1 gene expression and viral replication without sigdcant cytotoxicity. Partial thioation did not compromise its uptake, cellular distribution and nuclease resistance.

Mammalian ribonucleotide reductase is a highly regulated activity essential for DNA synthesis and repair. The 3′-untranslated region (3′-UTR) of mammalian ribonucleotide reductase R2 mRNA has been implicated in the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate-mediated stabilization of mouse BALB/c 3T3 R2 message. We investigated the possibility that the 3′-UTR contains regulatory information for R2 mRNA turnover. Using 3′-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3′-UTR a novel 9-nucleotide cis-element, 5′-UCGUGUGCU-3′, which interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) in a sequence-specific manner to form a 45-kDa R2 binding protein complex. The binding activity was redox-sensitive and down-regulated by 12-O-tetradecanoylphorbol-13-acetate and okadaic acid in a dose-dependent manner. Insertion of a 154-base pair fragment containing the cis-element led to markedly reduced accumulation of chloramphenicol acetyltransferase hybrid mRNA relative to the same insert carrying a series of G → A mutations within this element that eliminated binding. We suggest that the 9-nucleotide region functions as a destabilizing element. These results provide a model for ribonucleotide reductase gene expression through a novel and specific mRNA cis-trans-interaction involving a phosphorylation signal pathway that leads to changes in the stability of R2 message.

The hyaluronan (HA) receptor RHAMM is an important regulator of cell growth. Overexpression of RHAMM is transforming and is required for H-ras transformation. The molecular mechanism underlying growth control by RHAMM and other extracellular matrix receptors remains largely unknown. We report that soluble RHAMM induces G2/M arrest by suppressing the expression of Cdc2/Cyclin B1, a protein kinase complex essential for mitosis. Down-regulation of RHAMM by use of dominant negative mutants or antisense of mRNA also decreases Cdc2 protein levels. Suppression of Cdc2 occurs as a result of an increased rate of cdc2 mRNA degradation. Moreover, tumor cells treated with soluble RHAMM are unable to form lung metastases. Thus, we show that mitosis is directly linked to RHAMM through control of Cdc2 and Cyclin B1 expression. Failure to sustain levels of Cdc2 and Cyclin B1 proteins leads to cell cycle arrest.

Cell growth regulation by fibroblast growth factors (FGFs) is highly complex. The present study demonstrates a novel link between alterations in bFGF regulation during malignant conversion and the expression of ornithine decarboxylase, a key rate-limiting and regulatory activity in the biosynthesis of polyamines. H-ras transformed mouse 10T 1/2 cell lines exhibiting increasing malignant potential were investigated for possible bFGF-mediated changes in ornithine decarboxylase gene expression. Selective induction of ornithine decarboxylase gene expression was observed, since, in contrast to nontransformed 10T 1/2 cells and cells capable of only benign tumor formation, H-ras transformed metastatic cells exhibited marked elevations in ornithine decarboxylase message levels. Evidence for regulation of ornithine decarboxylase gene expression by bFGF at both transcription and posttranscription was found. Actinomycin D pretreatment of malignant cells prior to bFGF exposure inhibited the increase in ornithine decarboxylase message. Furthermore, striking differences in the rates of ornithine decarboxylase message decay were observed when cells treated with bFGF were compared to untreated control cells, with the half-life of ornithine decarboxylase mRNA increasing from 2.4 h in untreated cells to 12.5 h in cells exposed to bFGF. Evidence was also obtained for a cycloheximide-sensitive regulator of ornithine decarboxylase gene expression whose effect, in combination with bFGF, resulted in a further augmentation of ornithine decarboxylase gene expression. Furthermore, evidence is presented to suggest a possible role for G-protein-coupled events in the bFGF-mediated regulation of ornithine decarboxylase gene expression. The bFGF regulation of ornithine decarboxylase expression in H-ras transformed malignant cells appeared to occur independent of protein kinase C-mediated events. These results show that bFGF can modulate ornithine decarboxylase gene expression in malignant H-ras transformed cells and further suggests a mechanism of growth factor stimulation of malignant cells wherein early alterations in the regulatory control of ornithine decarboxylase gene expression are critical.

We compared the nucleosomal organization, histone H1 subtypes, and histone H1 phosphorylated isoforms of ras-transformed and parental 10T mouse fibroblasts. In agreement with previous studies, we found that ras-transformed mouse fibroblasts have a less condensed chromatin structure than normal fibroblasts. ras-transformed and parental 10T cells had similar amounts of H1 subtypes, proteins that have a key role in the compaction of chromatin. However, labeling studies with P and Western blot experiments with an antiphosphorylated H1 antibody show that interphase ras-transformed cells have higher levels of phosphorylated H1 isoforms than parental cells. G1/S phase-arrested ras-transformed cells had higher amounts of phosphorylated H1 than G1/S phase-arrested parental cells. Mouse fibroblasts transformed with fes, mos, raf, myc, or constitutively active mitogen-activated protein (MAP) kinase kinase had increased levels of phosphorylated H1. These observations suggest that increased phosphorylation of H1 is one of the consequences of the persistent activation of the mitogen-activated protein kinase signal transduction pathway. Indirect immunofluorescent studies show that phosphorylated H1b is localized in centers of RNA splicing in the nucleus, suggesting that this modified H1 subtype is complexed to transcriptionally active chromatin.

Ribonucleotide reductase is a key rate-limiting and regulatory step in DNA synthesis and plays a crucial role in the coordination of DNA synthesis, DNA repair, and cell proliferation. The present study demonstrates a link between alterations in TGF-beta 1 regulation during malignant conversion and the expression of ribonucleotide reductase. H-ras-transformed mouse 10T1/2 cell lines exhibiting malignant potential were examined for possible TGF-beta 1-mediated alterations in ribonucleotide reductase expression. Selective induction of ribonucleotide reductase gene expression occurred, since only H-ras-transformed highly metastatic cells exhibited marked elevations in ribonucleotide reductase expression, whereas nontransformed normal 10T1/2 cells were unaffected by TGF-beta 1 treatment. These changes occurred without any detectable modifications in DNA synthesis rates, suggesting that these changes were regulated by a novel mechanism independent of the S-phase of the cell cycle. Furthermore, this TGF-beta 1-mediated regulation of ribonucleotide reductase expression was shown to occur through an autocrine mechanism. TGF-beta 1-modulated regulation of ribonucleotide reductase expression requires de novo protein synthesis and involves, at least in part, transcriptional and post-transcriptional events. Furthermore, evidence is presented to suggest a possible role for protein kinase C-mediated events, protein phosphatases, and G-protein-coupled events in the TGF-beta 1-mediated regulation of ribonucleotide reductase expression in H-ras-transformed malignant cells. TGF-beta 1 regulation of ribonucleotide reductase in highly malignant cells appears to be complex and multifaceted and constitutes an integral part of an altered growth regulatory program.

Transforming growth factor (TGF-beta) is a family of multifunctional signalling molecules that play a fundamental role in both normal and malignant cell behavior. Procedures that alter mouse TGF-beta 1 gene expression provide an important approach for analyzing the complex regulatory processes associated with this member of the growth factor family. Therefore, we have designed oligodeoxyribonucleotides (oligos) in an antisense orientation, which are complementary to regions of the TGF-beta 1 message, in an attempt to obtain an oligo sequence that specifically reduces TGF-beta 1 synthesis. We observed that oligos containing a mixture of phosphorothioate and phosphodiester linkages were less toxic and more specific when compared to those only containing phosphorothioate. A non-toxic sequence was identified that markedly reduced the levels of TGF-beta 1 in oligo-treated malignant mouse fibrosarcoma cells. The invasive and metastatic properties of these fibrosarcoma cells were also significantly decreased following treatment with the antisense oligo. The results indicate an important role for altered TGF-beta 1 expression in the regulation of malignant cell proliferation, invasion and metastasis. These results also indicate that this oligo sequence is a useful tool for studies directed towards understanding the complex relationships between TGF-beta 1 and cellular regulation.

The human max protein has been shown to form heterodimers with myc family proteins. The ability of myc+max heterodimers to act as sequence-specific transcriptional activators appears to be essential for the oncogenic activity of myc. max (also called myn in murine cells) can homodimerize to form a transcriptionally inactive complex. We previously showed that in mouse 10T1/2 cells, a combination of activated ras, c-myc, and a mutant form of p53 can cooperate in the induction of cellular transformation and metastasis. In the study presented here, we tested the hypothesis that expression of the myn gene may play a role in this cooperative process. Analysis of myn mRNA in these cell lines revealed the presence of a major 2.0-kb RNA species. This message and the 21- and 22-kDa myn polypeptides it encodes were significantly over expressed in cells transformed by activated ras a lone, by ras in combination with c-myc or mutant p53, or by ras plus myc plus mutant p53, in comparison with untransformed parental 10T1/2 cells. We also found that induction of ras expression in a cell line harboring an inducible ras gene was accompanied by an increase in myn mRNA expression. Interestingly, cotransfection of 10T1/2 cells with ras and myn inhibited cellular transformation in a focus-forming assay when compared with transfection with ras alone. These results suggest a role for ras in the regulation of myn gene expression and suggest a model of oncogene cooperativity in which the relative levels of myc and myn gene expression can influence ras transformation of mouse 10T1/2 cells. © 1994 Wiley-Liss, Inc.

The R2 gene of ribonucleotide reductase is elevated in BALB/c 3T3 fibroblasts treated with the tumor promotor, 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment increased the half-life of the R2 message by 3-fold, showing that TPA regulates R2 gene expression by a post-transcriptional mechanism(s). A 20-nucleotide (nt) TPA-responsive region was found within the R2 mRNA 3'-untranslated region (3'UTR). Ultraviolet cross-linking detected a novel 45-kDa protein-R2 mRNA complex migration band that bound selectively to the 20-nt fragment and did not bind to the 5'UTR or the coding region of the R2 message, or to the 3'UTRs of mRNA from several other genes, or to the homopolymer poly(A) sequence. The-45 kDa protein-R2 mRNA binding activity observed in unstimulated cells was markedly down-regulated after TPA treatment. Deletion of a 201-nt region, containing the 20-nt sequence, from the 3'UTR caused stabilization of hybrid chloramphenicol acetyltransferase mRNA in the absence of TPA treatment. Furthermore, in vitro decay reaction mixtures supplemented with the 20-nt sense RNA transcript resulted in stabilization of R2 message. A model is presented of R2 message regulation in which a cis-element within the 20-nt sequence of the 3'UTR interacts with a cytosolic protein to form a 45-kDa protein-mRNA binding complex. The TPA-induced alteration of R2 message stability is at least in part due to the down-regulation of the 45-kDa protein-mRNA binding activity which is linked to a reduction in the rate of R2 mRNA degradation.

To assess the diagnostic accuracy of the H(2)S test for microbiological contamination of domestic water across different settings, as a basis for providing guidance on its use.We searched a range of bibliographic and 'grey' literature databases to identify studies that had processed domestic water samples using both the H(2)S test and recognized tests for thermotolerant coliforms or Escherichia coli. We screened 661 study abstracts and identified 51 relevant studies based on 13 853 water samples. For each relevant study, we recorded the level of correspondence between the H(2)S and recognized tests, microbial testing procedures, details of the samples processed and study quality indicators. We conducted a meta-analysis to investigate the impact of testing procedures, study quality and sample characteristics on the diagnostic accuracy of the H(2)S test.H(2)S test implementation varied between studies, and the test's diagnostic accuracy varied significantly and substantially between studies. Little of this variation was explained by testing procedures, study quality or the nature of the samples processed.Although in widespread use, our findings suggest that the diagnostic accuracy, particularly specificity, of the H(2)S test is variable. Optimal conditions for conducting the test remain unclear. As H(2)S test accuracy is low in a minority of these studies, we recommend that its performance be evaluated relative to standard methods, prior to its operational deployment in a new setting.

A rapid elevation of ribonucleotide reductase activity was observed with BALB c/3T3 fibroblasts treated with 10 nM okadaic acid, a nonphorbol ester tumor promoter and protein phosphatase inhibitor. Northern blot analysis of the two components of ribonucleotide reductase (R1 and R2) showed a marked elevation of R1 and R2 mRNA expression. Western blot analysis with R1 and R2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the elevation of the R2 rather than the R1 protein during treatment with okadaic acid. The okadaic acid induced elevations in R1 and R2 message levels occurred without a detectable change in the proportion of cells in S phase and were blocked by treatment of cells with actinomycin D, indicating the importance of the reductase transcriptional process in responding to the action of okadaic acid. Furthermore, down-regulation of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate pretreatment abrogated the okadaic acid mediated elevation of ribonucleotide reductase mRNAs, consistent with the involvement of this signal pathway in the regulation of ribonucleotide reductase and the effects of okadaic acid. Treatment of cells with 2.5 nM calyculin A, another non-phorbol ester tumor promoter and protein phosphatase inhibitor, resulted in a rapid elevation of both R1 and R2 mRNA levels within 10 min of treatment. This first demonstration that the non-phorbol ester tumor promoters and protein phosphatase inhibitors can cause rapid alterations in ribonucleotide reductase gene expression suggests that (i) ribonucleotide reductase, particularly the R2 component, plays a fundamental role in the critical early events involved in the process of tumor promotion, and (ii) illustrates a role for cellular protein phosphatases in the regulation of ribonucleotide reductase and, through this process, the regulation of DNA synthesis.Key words: ribonucleotide reductase, DNA synthesis, okadaic acid, calyculin A, tumor promoter, protein phosphatase.

Ribonucleotide reductase provides the four deoxyribonucleotides required for the synthesis of DNA. In this study, we examined the hypothesis that alterations in the regulation of ribonucleotide reductase activity may be necessary to provide the deoxyribonucleotides required for DNA repair, following exposure of mammalian cells to DNA damaging agents such as the antitumor agent chlorambucil. We observed a marked transient increase in ribonucleotide reductase activity within 2 h of exposing BALB/c 3T3 mouse cells to DNA damaging concentrations of chlorambucil. Northern blot analysis showed that elevations in activity were accompanied by transient increases in the mRNA levels of both genes (R1 and R2) that code for ribonucleotide reductase. Western blot analysis indicated that only the protein for the limiting component for enzyme activity, R2, was significantly elevated in chlorambucil treated cultures. The chlorambucil effects upon activity and regulation of ribonucleotide reductase occurred without any detectable changes in the rate of DNA synthesis, as would be expected if the elevation in enzyme activity is required for DNA repair. The chlorambucil-induced elevations in R1 and R2 message levels were blocked by treatment of cells with actinomycin D or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, indicating the importance of the reductase transcriptional process in responding to the action of chlorambucil and providing evidence for the involvement of a protein kinase C pathway in the regulation of mammalian ribonucleotide reductase. In addition to the chlorambucil-induced elevations in enzyme activity, message, and protein levels, the drug was also shown to be an inhibitor of ribonucleotide reductase activity in cell-free preparations. Separation of ribonucleotide components on an affinity column followed by selective exposure of the protein components to chlorambucil showed that both R1 and R2 proteins were targets for chlorambucil, in keeping with the known alkylating abilities of the drug. These observations provide the first direct demonstration of a link between the regulation of mammalian ribonucleotide reductase and the process of DNA repair and contribute to our understanding of the mode of action of a class of drugs represented by chlorambucil, in which chemotherapeutic activity has been attributed to DNA damaging effects.

Previous investigations have indicated that the suppression of proliferation by transforming growth factor (TGF) beta 1 is often lost upon cellular transformation, and that proliferation of some tumors is stimulated by TGF-beta. The present study provides the first observation of a link between TGF-beta 1 regulation of this process and alterations in the expression of ribonucleotide reductase, a highly controlled rate-limiting step in DNA synthesis. A series of radiation and T24-H-ras-transformed mouse 10T1/2 cell lines exhibiting increasing malignant potential was evaluated for TGF-beta 1 induced alterations in ribonucleotide reductase M1 and M2 gene expression. Early increases in M1 and/or M2 message and protein levels were observed only in malignant cell lines. The TGF-beta 1 induced changes in M1 and/or M2 gene expression occurred prior to any detectable changes in the rates of DNA synthesis, supporting the novel concept that ribonucleotide reductase gene expression can be elevated by TGF-beta 1 without altering the proportion of cells in S phase. T24-H-ras-transformed 10T1/2 cells were transfected with a plasmid containing the coding region of TGF-beta 1 under the control of a zinc-sensitive metallothionein promoter. When these cells were cultured in the presence of zinc, a large induction of TGF-beta 1 message was observed within 1 h. Both M1 and M2 genes were also induced, with increased mRNA levels appearing 2 h after zinc treatment, or 1 h after TGF-beta 1 message levels were clearly elevated. In total, the data suggests a mechanism of autocrine stimulation of malignant cells by TGF-beta 1, in which early alterations in the regulation of ribonucleotide reductase may play an important role.

Highly metastatic mouse 10T1/2 cell lines (Ciras 2, Ciras 3 and dGC2M5) which have been T24-H-ras transfected, are shown to have differential responses in metastatic properties when grown in the presence of the processing inhibitors, swainsonine, castanospermine and deoxymannojirimycin. Concanavalin A binding data indicated the inhibitors caused similar shifts in oligo-saccharide structures, resulting in more high mannose character for all cell lines. However, swainsonine inhibited the experimental metastasis of dGC2M5, but did not affect the metastatic properties of Ciras 2 and Ciras 3. Inversely, castanospermine reduced experimental metastasis of Ciras 2 and 3 and did not inhibit dGC2M5. These results show that closely related metastatic cell lines respond differently in their metastatic ability when changes occur in N-linked oligosaccharide content. This observation emphasizes the importance of oligosaccharide structure in the malignant phenotype and indicates that some caution should be used when generalizing about the effects of processing inhibitors on a complex process like metastasis.

Hydroxyurea was used to select two very highly drug resistant cell lines, designated HR-15 and HR-30. Both drug resistant lines contained elevated levels of ribonucleotide reductase activity. Northern and Southern blot analysis indicated that the two drug resistant lines contained increased levels of mRNA for the two components, M1 and M2, of ribonucleotide reductase, and M1 and M2 gene amplifications. Alterations in M1 and M2 protein levels were also evident in Western blot analysis. Further studies of HR-15 and HR-30 cells by Northern and Southern blot analysis showed that the drug resistant cell lines had elevated levels of ornithine decarboxylase mRNA and p5-8 mRNA, as well as increased ornithine decarboxylase and p5-8 gene copy numbers, respectively. Furthermore, characterization of HR-15 and HR-30 drug-resistant cell lines revealed increased mRNA levels for both H- and L-ferritin. Both cell lines exhibited by Southern blot analysis, amplification of the H- and L-ferritin genes. Increases in the cellular levels of H- and L-ferritin subunit proteins were also observed in both HR-15 and HR-30 cells, by Western blot analysis. This is the first description of mutant cell lines containing this complex combination of modified gene expressions and gene amplifications. The alterations exhibited by these lines confirm and extend present models of hydroxyurea resistance, are in agreement with and help substantiate models of ribonucleotide reductase regulation and provide interesting links between the expressions of several cellular activities important in proliferation.

Secreted phosphoprotein I (SPP), also known as 2ar, osteopontin, 44-kDa bone phosphoprotein, bone sialoprotein I, and transformation-related phosphoprotein, is a 41.5-kDa glycosylated phosphoprotein secreted by many mammalian cell lines and expressed in a limited set of tissues. Using a cDNA probe, we found that SPP mRNA, which is barely detectable in normal mouse epidermis, was expressed at moderate-to-high levels in 2 of 3 epidermal papillomas and at consistently high levels in 7 of 7 squamous-cell carcinomas induced by an initiation-promotion regimen. This contrasts with the transient induction we had previously observed after a single application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). In a set of 5 independently isolated T24-H-ras-transfected mouse C3H 10T1/2 cell lines, the levels of SPP mRNA correlated well with ras mRNA levels and with both experimental and spontaneous metastatic ability. SPP mRNA expression was also elevated in a derivative of mouse LTA cells transfected with genomic DNA from B16F1 melanoma cells and selected for increased experimental metastatic ability in the chick embryo. This apparent association of SPP expression with invasion, progression and metastasis, along with the presence of a functional ArgGlyAsp (RGD) cell adhesion site in SPP (osteopontin), leads us to propose that SPP may act as an autocrine adhesion factor for tumor cells.

We have examined the effects of transformation by activated H-ras and other transforming oncogenes on the activity of the enzyme, heparanase. Degradation of 3H-N-acetylated-partially N-desulfated heparan sulfate by cellular extracts of the transformants was assessed by gel permation chromatography. More extensive degradation was observed with 10T1/2 mouse embryo fibroblasts transfected with an activated H-ras oncogene. The cells having the highest metastatic potential (CIRAS-3) were shown to contain the greatest heparanase activity, giving 49% higher levels of activity than parental cells (P less than 0.0002). Furthermore, the enzyme activity produced by a series of H-ras transformed cell lines increased progressively with metastatic potential (non-parametric rank correlation coefficient r = 0.96). Transfection of NIH 3T3 fibroblasts with activated H-ras, v-src or v-fes oncogenes, which induced the metastatic phenotype, did not lead to large increases in heparanase activities. Also, inhibition of ras-induced malignancy by cotransfection of rat REF cells with the Ad2 E1a oncogene did not produce significant declines in heparanase activities. These results are consistent with the view that modifications in heparanase activity can play a role in the complex process of metastasis in some, but not all situations.

In the present study, we show that hydroxyurea-inactivated ribonucleotide reductase protein M2 has a destabilized iron center, which readily releases iron. In addition, evidence is presented which indicates that single or multistep selection for hydroxyurea resistance, in a variety of mammalian cell lines, leads to alterations in the expression of the gene for the iron storage protein, ferritin. In all hydroxyurea-resistant cell lines examined, including human, hamster, rat, and mouse, there was an elevation in ferritin heavy (H)- and/or light (L)-mRNA levels, but no change in the corresponding gene copy number. A detailed analysis of ferritin expression in a hydroxyurea-resistant mouse L cell line showed that when compared to its wild type counterpart, there was an increase in H subunit concentration but no significant change in L subunit levels. The increased H/L subunit ratio was not brought about by specific changes in the rates of ferritin subunit biosynthesis, but rather resulted from changes in the post-translational stability of H subunits relative to L subunits in the resistant cell line compared to its parental wild type. Also, we show that treatment of cells with hydroxyurea results in an increased rate of ferritin biosynthesis in the absence of changes in H- or L-mRNA levels. These results indicate that the development of even low level hydroxyurea resistance in mammalian cells may require alterations in ferritin gene expression, and they show an interesting relationship between the expressions of two highly regulated activities, ribonucleotide reductase and ferritin.

Three types of normal human cells were selected in tissue culture with three drugs without observing a single amplification event from a total of 5 x 10(8) cells. No drug-resistant colonies were observed when normal foreskin keratinocytes were selected with N-(phosphonacetyl)-L-aspartate or with hydroxyurea or when normal mammary epithelial cells were selected with methotrexate. Some slightly resistant colonies with limited potential for growth were obtained when normal diploid fibroblast cells derived from fetal lung were selected with methotrexate or hydroxyurea but careful copy-number analysis of the dihydrofolate reductase and ribonucleotide reductase genes revealed no evidence of amplification. The rarity of DNA amplification in normal human cells contrasts strongly with the situation in tumors and in established cell lines, where amplification of oncogenes and of genes mediating drug resistance is frequent. The results suggest that tumors and cell lines have acquired the abnormal ability to amplify DNA with high frequency.

The Johns Hopkins University Applied Physics Laboratory is developing a technique that uses aggregate system performance data to explore multi-dimensional trade spaces among military capabilities. The process is used to examine the operational value of trading systems within capabilities and capabilities within warfare areas. The methodology models joint military performance and examines capabilities associated with combining systems. Evaluation of tactics, techniques and procedures, measures of performance, and measures of outcome is common within the Department of Defense; developing an over-arching predictive tool is desired. Trade space analysis, response surface methodology, and optimization are not new approaches; the combination of these into an orchestrated process is. A variety of capabilities each with a range of performance values combine to produce results in an area. A specific capability determines the end state resulting of combination; the desired end states may differ from campaign to campaign. All possible combinations of contributing systems describe the capability. An optimization process explores the performance parameters for all systems simultaneously, aggregates system values to define capabilities, and searches for the optimal combination of capabilities. The optimization process is provided response surface approximations over which to search for optimal regions. (14 refs.)

Ribonucleotide reductase is an enzyme that is essential for DNA synthesis and repair. It is composed of 2 dimeric proteins called R1 and R2 that are both necessary for enzymatic activity that reduces ribonucleotides to deoxyribonucleotides. This is the rate-limiting reaction that provides a supply of precursors for DNA synthesis therefore it is essential for cell proliferation. The importance of understanding the complex regulation of ribonucleotide reductase is emphasized by observations that mechanisms controlling its expression and activity may be altered during malignant cell proliferation which leads to drug resistance, making it a useful target to develop chemotherapeutic compounds in the treatment of cancer. Expression studies with the R1 and R2 genes have provided evidence for a direct role for the components of ribonucleotide reductase in determining malignant potential. Ribonucleotide reductase is regulated by transcriptional activation of gene expression and post-transcriptional mechanisms that alter mRNA message stability. Post-transcriptional regulation of mRNA turnover plays an important role in modulating mRNA steady state levels and therefore directly influences gene expression. The 3'-untranslated region (UTR) of R1 and R2 messages contain sequences that are important in regulating gene expression through changes in message stability. Studies have found that mRNA message stability is mediated by growth factors, cytokines and tumor promoters. Several studies have elucidated signal transduction pathways of tumor promoters, TGF-beta and oxidation/reduction agents. This report reviews how knowledge of these signaling pathways is revealing new insights into how ribonucleotide reductase mRNA binding proteins are important in regulating cellular proliferation, drug resistance and malignancy.

Here, we describe a gentle and effective method for the rapid and reproducible isolation of histone-bound extrachromosomal DNA molecules called extrachromosomal elements (EEs). This method facilitates the harvest of a specific population of EEs following their isolation from cultured cells, primary tissues, and tumor cells. Active EEs are bound to histone proteins, and these histone-bound EEs carry actively transcribing genes such as c-myc. Our method exploits the presence of histones on EEs and serves as a first-step purification procedure, allowing for the cloning or multivariant analysis of an immunopurified sample of EEs. We isolated EEs from 4-hydroxytamoxifen (4-HT)-activated Myc-ER™-regulatable Pre-B ABM cells. Following one round of immunoprecipitation, we demonstrate the purification of histone-bound EEs. We confirmed that our purification enriched for EEs that carry genes by fluorescent in situ hybridization of EEs (FISH-EEs), and we probed non-enriched and immunopurified EEs with a dihydrofolate reductase (DHFR) cDNA probe that is known to detect extrachromosomal amplification in Myc-activated cells. We demonstrate the enrichment of immunoprecipitated DHFR-containing extrachromosomal DNA molecules.

We have previously shown that mouse phosphorylated histone H1b (pH1b) was localized near nuclear sites that contained splicing factors. This observation suggested to us that pH1b was associated with transcribing chromatin. Here we investigated the relationship between phosphorylation of H1b and transcription. We demonstrate that treatment of normal or ras-transformed mouse fibroblasts with the transcription inhibitor actinomycin D for 70 min results in a dramatic decrease in the level of pH1b. Similar results were observed when transcription was inhibited by 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB). When DRB was removed, the level of pH1b was restored after 2 h. Treatment of the cells with aphidicolin, a potent inhibitor of replication, resulted in a marked decrease in the level of pH1b after 30 min. This is the first report showing a dependence of histone modification upon ongoing transcription and replication. Inhibition of transcription or replication may hinder accessibility of H1b to the H1 kinase, supporting the idea that pH1b is associated with transcribing chromatin. Phosphorylation of H1b may be required to maintain a more decondensed chromatin structure to facilitate transcription and replication processes.

GPI2A is a 20-mer antisense oligonucleotide sequence that is complementary to a region of the HIV-1gaggene. An analysis of viral core antigen p24 protein synthesis inhibition was performed with cells expressing HIV-1 proteins, following treatment with GPI2A or eight other unique antisense constructs designed to bind to regions of thegaggene, at positions that 5′ or 3′ flank the GPI2A target site. GPI2A was found to be the most effective construct, indicating that the GPI2A target region is a particularly sensitive site for antisense activity. An analysis of energy-related parameters important in complementary duplex formation was performed for each antisense construct. Also, the potential of each antisense sequence to exhibit self-complementarity or to self-dimerize was assessed. The results from these analyses provided an explanation for the high specificity and the superior inhibitory characteristics of GPIA when compared to the eight other antisense oligonucleotides. GPI2A exhibited the second most favorable energy-related characteristics for hybridization reactions, and most importantly, unlike the other eight antisense sequences, it did not show the potential to self-complement or to dimerize. The results of this study and a previous investigation of sequence specificity requirements for GPI2A inhibition of HIV-1 gene expression provide strong evidence for an antisense mode of action for this oligonucleotide construct, a useful tool for analysis of viral gene expression and perhaps a potential therapeutic agent.

The R2 component of ribonucleotide reductase is rate-limiting for DNA synthesis in proliferating cells, and recently, it has been shown that aberrant expression of R2 directly alters the malignant potential of tumor cells. We show that R2 gene expression is elevated in BALB/c 3T3 cells treated with transforming growth factor (TGF)-beta 1, TGF-beta 2, or TGF-beta 3, as determined by Northern blot analysis. Gel shift assays and UV crosslinking studies demonstrated similar post-transcriptional regulation at the 3'-untranslated region (3'-UTR) of the R2 mRNA, by TGF-beta 1, TGF-beta 2, and TGF-beta 3. The three growth factors induced a common 75 kDa RNA-protein complex. A 9 nucleotide sequence, GAGUUUGAG, previously shown to be responsive to TGF-beta 1-mediated R2 message stability changes, effectively competed out the formation of the R2 3'-UTR complex. We propose that these three different members of the TGF-beta family work through a common mechanism to control an important component of cell proliferation and a potential determinant of malignant progression.

The receptor for hyaluronan mediated motility (RHAMM) gene expression is markedly elevated in fibrosarcomas exposed to transforming growth factor-beta1 (TGF-beta1). The half-life of RHAMM mRNA was increased by 3 fold in cells treated with TGF-beta1, indicating that growth factor regulation of RHAMM gene expression at least in part involves a posttranscriptional mechanism. Our studies demonstrated that a unique 30-nucleotide (nt) region that has three copies of the sequence, GCUUGC, was the TGF-beta1-responsive region in the 3'-untranslated region (3'-UTR) that mediated message stability. This region interacted specifically with cytoplasmic trans-factors to form multiple protein complexes of approximately 175, 97, 63, 26, and 17 kDa post-TGF-beta1 treatment, suggesting a role for these complexes in the mechanism of action of TGF-beta1-induced message stabilization. Insertion of the 3'-UTR into the chloramphenicol acetyltransferase gene conferred TGF-beta1 induced stability of chloramphenicol acetyltransferase-hybrid RNA in stably transfected cells, while the same insert carrying a deletion containing the 30-nt region had no significant effect on mRNA stability. These results provide a model of RHAMM message regulation in which TGF-beta1-mediated alteration of RHAMM message stability involves the up-regulation of multiple protein interactions with a 30-nt cis-element stability determinant in the 3'-UTR. This model also suggests that this 30-nt base region functions in cis to destabilize RHAMM mRNA in resting normal cells.

H1 histones play an important role in regulating higher order structure of chromatin and are potential regulators of gene expression. H1s are phosphorylated, a modification which alters their interaction with DNA. We measured the abundance of three phosphorylated H1 subtypes in mouse fibroblasts transformed by combinations of ras, myc and mutant p53 which differ in metastatic potential. We found that there is an increase in phosphorylation of H1 subtypes in fibroblasts transformed with ras, myc and mutant p53. This increase was found to correlate with cellular transformation but not with induction of the metastatic phenotype.

Ribonucleotide reductase R2 gene expression is elevated in BALB/c 3T3 fibroblasts treated with transforming growth factor beta 1. We investigated the possibility that the 3'-UTR of ribonucleotide reductase R2 mRNA contains regulatory information for TGF-beta 1 induced message stability. Using end-labeled RNA fragments in gel shift assays and UV cross-linking analyses, we detected in the 3'-UTR a novel 9 nucleotide (nt) cis element, 5'-GAGUUUGAG-3' site, which interacted specifically with a cytosolic protease sensitive factor to form a 75 kDa complex. The cis element protein binding activity was inducible and markedly up-regulated cross-link 4 h after TGF-beta 1 treatment of mouse BALB/c 3T3 cells. Other 3'-UTRs [IRE, GM-CSF, c-myc and homopolymer (U)] were poor competitors to the cis element with regard to forming the TGF-beta 1 dependent RNA-protein complex. However, the cis element effectively competed out the formation of the R2 3'-UTR protein complex. Cytosolic extracts from a variety of mammalian cell lines (monkey Cos7, several mouse fibrosarcomas and human HeLa S3) demonstrated similar TGF-beta 1 dependent RNA-protein band shifts as cell extract from BALB/c 3T3 mouse fibroblasts. Binding was completely prevented by several different mutations within the cis element, and by substitution mutagenesis, we were able to predict the consensus sequences, 5'-GAGUUUNNN-3' and 5'-NNNUUUGAG-3' for optimal protein binding. These results support a model in which the 9 nt region functions in cis to destabilize R2 mRNA in cells; and upon activation, a TGF-beta 1 responsive protein is induced and interacts with the 9 nt cis element in a mechanism that leads to stabilization of the mRNA. This appears to be the first example of a mRNA binding site that is involved in TGF-beta 1-mediated effects.

Previous studies have demonstrated that oligodeoxynucleotide phosphorothioates complementary to human immunodeficiency virus type 1 (HIV-1) RNA are more nuclease resistant and are effective inhibitors of HIV-1 replication than their unmodified counterpart. In this study, antisense oligodeoxynucleotide sequences were evaluated for therapeutic potential in the treatment of HIV infections. The use of HIV-infected lymphocytes to test the efficacy of a drug is very complex, and therefore it is difficult to draw conclusions about the mechanism. We used a COS-like Monkey kidney cell line (CMT3) stably transfected with plasmids pCMVgagpol-rre-r (containing gag and pol genes) and pCMVrev (containing the rev gene of HIV-1), derived from cDNA clone BH10, as a model. A biologically active provirus that transcribes and translates their nucleotide sequences into viral proteins p24, p39/41, p55, and p160 was generated. Sequence-specific and dose-dependent inhibition of HIV-1 viral protein synthesis and significant inhibition at the mRNA level were demonstrated by antisense construct GPI2A, directed against a nonregulatory region of the HIV-1 genome. Also, our studies demonstrated enhancement of the antisense effect through encapsulation in a cationic lipid preparation. The observed attenuation of HIV-1 mRNA levels suggests that, at least in part, the mechanism of action of GPI2A was at the transcript level. Further studies have also shown antiviral activity of this construct as determined by the reverse transcriptase assay using acutely and chronically infected cells of lymphoid origin (H9 cells). Toxicological studies involving cell growth characteristics, colony-forming ability, effects on cellular proteins, specific activities of labeled proteins, and DNA synthesis in cell culture showed no cytotoxic effects of GPI2A.

Ribonucleotide reductase is a highly regulated rate-limiting enzyme activity in DNA synthesis, responsible for reducing ribonucleotides to their deoxyribonucleotide forms. Using 3'-end labeled RNA and band-shift and UV cross-linking analyses, we have identified a cis-element, 5'-CAAACUUC-3', within the 3'-untranslated region of the mammalian ribonucleotide reductase R1 mRNA, which binds a cytoplasmic protein in BALB/c 3T3 mouse cells, to form a 57 kDa RNA-protein complex. Sequence-specific binding was observed, and binding was prevented by several different mutations within the cis-element. We suggest that this cis-trans interaction plays a role in R1 mRNA stability.

We have tested the hypothesis that H-ras transformed cells contain alterations in signal pathways important in controlling the expression of ornithine decarboxylase (ODC), the highly regulated rate-limiting activity in the biosynthesis polyamines. Mouse 10 T 1/2 fibroblasts and a series of 10 T 1/2 H-ras transformed cell lines were treated with stimulators of cAMP synthesis (forskolin and cholera toxin), a biologically stable analogue of cAMP (8-bromo-cAMP), and an inhibitor of cAMP degradation (3-isobutyl-1-methylxanthine). Elevations in ODC gene expression were noted in H-ras transformed cells that were not observed in parental 10 T 1/2 fibroblasts. The forskolin-mediated effects were not detected with 1,9-dideoxyforskolin, a compound structurally related to forskolin, which does not activate adenyl cyclase. The effects observed with cholera toxin were not detected when cells were treated with the purified subunits of this compound, indicating that the toxin-induced effects were cAMP-specific. Actinomycin D treatment prior to forskolin exposure reduced the elevation observed in ODC gene expression indicating the involvement of the transcriptional process. Furthermore, we observed that cycloheximide treatment of malignant but not benign H-ras transformed cells significantly elevated ODC message level. Treatment of malignant cells with both cycloheximide and forskolin together resulted in a further additive elevation in ODC message, but a similar treatment of benign tumor cells reduced the forskolin-mediated increase in ODC message. In addition, treatment of H-ras transformed cell with the tumor promoter, 12-0-tetradecanoylphorbol-13-acetate (TPA) led to an elevation in ODC mRNA levels not observed in parental 10 T 1/2 fibroblasts. Cells treated with a combination of TPA and forskolin. Exposure of H-ras transformed cells to TPA and forskolin together led to further pronounced increases in ODC message when compared to treatment with TPA or forskolin alone. These results demonstrate aberrant regulation of signal pathways involved in controlling ODC gene expression in H-ras transformed cells and provide further insight into the altered growth regulatory program associated with malignant transformation. © 1994 Wiley-Liss, Inc.

We have investigated the genetic stability of NIH-3T3 cells transfected with sequences coding for basic fibroblast growth factor (bFGF) by determining drug resistance and gene amplification potential. Colony-forming experiments and fluctuation analyses showed that the frequency and rate of resistance to N -(phosphonacetyl)-L-aspartate (PALA) was dramatically elevated in cells transfected with either the normal bFGF coding sequence that lacks a known signal for secretion or a chimeric bFGF sequence that targets the growth factor to the secretory pathway. Basic FGF-transfected cells that grew in the presence of PALA were found to possess an amplification of the CAD gene, which codes for a multifunctional protein involved in pyrimidine biosynthesis and is the site of action for PALA. The observation that these alterations occur in cells transfected with a bFGF sequence, without a conventional signal sequence for secretion, suggests an intracrine as opposed to autocrine mechanism of action. The results describe a new function for this growth factor and suggest a novel role for aberrant expression of bFGF in mechanisms of tumor progression.