Insulin like growth factor-1 selectively regulates the expression of matrix metalloproteinase-2 in malignant H-ras transformed cells.

The present study demonstrates alterations in the regulation of matrix metalloproteinase-2 (MMP-2) expression in response to insulin like growth factor-1 (IGF-1) in a H-ras transformed cell line, C3, which is capable of metastasis formation. These changes in MMP-2 expression in response to IGF-1 treatment did not occur in either non-transformed parental 10 T 1/2 cells or in H-ras transformed cells (NR3 cells) which are capable of benign tumour formation. IGF-1 treatment of C3 cells resulted in increased expression of MMP-2 gelatinolytic activity and increased expression of MMP-2 mRNA levels. The IGF-1 mediated alterations in MMP-2 mRNA levels were dependent upon de novo protein synthesis and independent of transcriptional events, but dependent upon post-transcriptional regulatory events. Most notably, IGF-1 can regulate MMP-2 mRNA expression in C3 cells through a mechanism involving MMP-2 message stabilization. This study demonstrates aspects of the temporal regulatory mechanisms of MMP-2 expression in response to insulin-like growth factor-1 in a H-ras transformed fibrosarcoma cell line capable of metastasis formation and thereby, provides further insight into the altered growth regulatory program associated with H-ras mediated cellular transformation and malignant progression.

S-Adenosylmethionine decarboxylase gene expression is regulated by the cAMP signal transduction pathway in H-ras transformed fibrosarcoma cells capable of malignant progression.

The hypothesis that H-ras transformed cells contain alterations in signalling pathways important in controlling the expression of S-adenosylmethionine decarboxylase, (SAMDC) a highly regulated activity in the biosynthesis of polyamines was tested. Mouse 10 T1/2 fibroblasts and H-ras transformed cell lines of varying degrees of malignant potential were treated with agents which affect cAMP levels within cells. Elevations in SAMDC expression were noted in H-ras transformed metastatic C3 cells, which were not observed in either parental, non-transformed 10 T1/2 fibroblast cells, or in ras transformed NR3 cells, which are only capable of benign tumour formation. Forskolin, a stimulator of cAMP synthesis, was able to increase SAMDC enzyme activity but the response which occurred was dependent upon the cellular phenotype expressed. Actinomycin D pre-treatment of C3 cells prior to exposure to forskolin did not abrogate the elevation observed in SAMDC gene expression suggesting that this was not a transcriptional process mediated event. Forskolin pre-treatment of C3 cells did result in a marked increase in the half-life of SAMDC mRNA transcripts suggesting a role for post-transcriptional stabilization. Furthermore, cycloheximide treatment of malignant C3 cells resulted in elevated SAMDC mRNA levels. Treatment of malignant C3 cells with both cycloheximide and forskolin together resulted in a further additive elevation in SAMDC message levels. Cycloheximide treatment alone was found to affect the half-life of SAMDC mRNA through a mechanism of post-transcriptional stabilization. Additionally, altered SAMDC gene expression in C3 cells which occurred in response to cAMP alterations, was enhanced by stimulation of a protein kinase C pathway suggesting possible interactions between protein kinase C-and cAMP-mediated pathways which affect the regulation of SAMDC expression in highly malignant C3 cells. These results demonstrate aberrant regulation of signalling pathways involved in controlling SAMDC gene expression in H-ras transformed cells capable of malignant progression and provide further insight into the altered growth regulatory program associated with H-ras mediated cellular transformation and malignant progression. J. Cell. Biochem. Suppl. 36: 209-221, 2001.

Phorbol ester tumour promoter mediated altered expression and regulation of matrix metalloproteinase-2 in a H-ras transformed cell line capable of benign tumour formation.

The matrix metalloproteinases (MMPs) are thought to play key roles in tumour formation and malignant progression. The present study demonstrates alterations in the regulation of matrix metalloproteinase-2 (MMP-2) expression in response to the phorbol ester tumour promoter, PMA, in a H-ras transformed cell line, NR3, which is capable of benign tumour formation. PMA treatment of NR3 cells resulted in decreased expression of MMP-2 mRNA levels. Following a lag period, an accompanying change in gelatinolytic activity was also found. These PMA-mediated alterations in MMP-2 mRNA levels were independent of de novo protein synthesis and involved both transcriptional and post-transcriptional events. Most notably, PMA regulates MMP-2 mRNA expression through a mechanism involving message de-stabilization. Additionally, protein kinase C mediated events were found to play a role(s) in the regulation of MMP-2 message expression in NR3 cells. This study demonstrates several novel aspects regarding the regulation of MMP-2 expression in a H-ras transformed cell line and thereby provides further insight into the altered growth regulatory programs associated with H-ras mediated cellular transformation.

Platelet-derived growth factor mediated altered expression and regulation of ornithine decarboxylase in H-ras-transformed cell lines.

This study demonstrates a novel link between alterations in platelet-derived growth factor (PDGF) regulation of ornithine decarboxylase (ODC) expression during malignant conversion. H-ras-transformed cell lines exhibited PDGF-mediated alterations in ODC gene expression. These alterations involved transcriptional, posttranscriptional, and cycloheximide-mediated events. PDGF-mediated alterations in ODC gene expression in NR3 cells (capable of only benign tumour formation) were ras-dependent, involved a tyrosine kinase activity and mitogen-activated protein (MAP) kinase-mediated signalling events, and were independent of both protein kinase C (PKC) events and pertussis toxin-sensitive (PTS) G-protein-mediated signalling. PDGF-mediated alterations in ODC gene expression in C2 cells [capable of malignant progression (metastasis formation)] were ras-dependent, required a tyrosine kinase activity, involved both MAP kinase-mediated events and phosphatidylinositol-3-kinase (PI-3-kinase)-mediated events, and were dependent upon PTS G-protein-mediated signalling but independent of PKC-mediated events. PDGF-mediated regulation of ODC gene expression changes in response to H-ras-mediated cellular transformation and malignant progression.

Altered matrix metalloproteinase expression associated with oncogene-mediated cellular transformation and metastasis formation.

Matrix metalloproteinase expression was examined in a series of mammalian cell lines of varying degrees of malignant progression. The expression of MMP-2 and MMP-9 was found to correlate with ras-mediated cellular transformation and as a function of malignant potential. Altered MMP-2 and MMP-9 expression was found to correlate also in other oncogene transformed cell lines and the level of expression of both MMP-2 and MMP-9 correlated with metastatic potential. Increased expression of both MMP-2 and MMP-9 was also found in cells which constitutively over-express MAP kinase kinase suggesting that one of the consequences of the persistent activation of the MAP kinase pathway is elevated expression of MMP-2 and MMP-9. Additionally, this study demonstrated a correlation between the expression of MMP-3 (stromelysin-1) and the level of ras expressed in cells and with the cells' ability to form tumors and with malignant potential. The existence of a novel 80 kDa caseinase activity which correlates with ras expression and the ability of the cell to form tumors was also demonstrated. The growth status of transformed cells was also found to be important in determining the expression of MMP-2 mRNA but not MMP-9 mRNA expression, and this expression was cell-type specific. This study also demonstrates that oncogenes can interact to influence and to determine the nature of the matrix metalloproteinases expressed and that this interaction results in a tumorigenic phenotype and, most importantly, contributes to the metastatic phenotype. Alterations in the expression and the regulation of MMPs, particularly MMP-2 and MMP-9, constitute an integral part of the altered growth regulatory program found within transformed cells and in particular, in transformed cells capable of malignant progression.

Tumour promoter mediated altered expression and regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase in H-ras-transformed fibrosarcoma cell lines.

Alterations in cellular growth are important in the progression of malignant disease. Cell growth regulation by tumour promoters can be complex. The present study demonstrates a novel link between alterations in phorbol ester tumour promoter mediated regulation during malignant conversion and the expression of ornithine decarboxylase and S-adenosylmethionine decarboxylase, key rate-limiting and regulatory activities in the biosynthesis of polyamines. H-ras-transformed mouse 10 T 1/2 cell lines exhibiting increasing malignant potential were investigated for possible phorbol ester tumour promoter mediated changes in ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) gene expression. Selective induction of ODC and SAMDC gene expression was observed, since in contrast to nontransformed parental 10 T1/2 cells, ras-transformed cells capable of benign tumour formation (NR3 cells) and ras-transformed cells capable of metastasis formation (C2 cells) exhibited marked alterations in the levels of ODC and SAMDC gene expression. Increased ODC gene and SAMDC gene expression in response to phorbol-12-myristate-13-acetate (PMA) treatment was found to involve transcriptional events in both NR3 cells and in C2 cells. Post-transcriptional events also played a role in the regulation of ODC gene expression in NR3 cells and in C2 cells, and in the regulation of SAMDC gene expression in C2 cells but not in NR3 cells. In NR3 cells, alterations in ODC and in SAMDC gene expression was an event requiring de novo protein synthesis, whereas in highly malignant C2 cells, protein synthesis inhibition following cycloheximide treatment in cooperation with PMA resulted in an augmentation of both ODC and SAMDC gene expression. Evidence is presented to suggest that the PMA-mediated alterations in ODC and in SAMDC gene expression in NR3 cells and in C2 cells involved protein kinase C - mediated events. The status of the cellular polyamine levels was also an important determinant of the PMA-mediated alterations that occurred in ODC and in SAMDC expression in these H-ras transformed cells. Collectively, these results suggest that PMA can modulate ODC and SAMDC expression in H-ras transformed cells and that the mechanisms involved in the PMA- mediated regulation of ODC and SAMDC gene expression changes as a function of H-ras mediated cellular transformation and malignant progression. This study further suggests a mechanism of PMA stimulation of transformed cells wherein early alterations in the regulatory control of ODC and SAMDC gene expression are important and critical.

Transformation by H-ras can result in aberrant regulation of ornithine decarboxylase gene expression by transforming growth factor-beta(1).

Inhibition of DNA synthesis and cell proliferation is frequently lost during malignant transformation and occasionally, tumour cell proliferation is actually stimulated by transforming growth factor beta(1) (TGF-beta(1)). The present study demonstrates a novel link between alterations in TGF-beta(1) regulation during cellular transformation and malignant conversion and the expression of ornithine decarboxylase (ODC) which is a key rate limiting activity in the biosynthesis of polyamines and which is an enzyme that plays an important role in cell growth and differentiation. H-ras transformed mouse 10T(1/2) cell lines of varying degrees of malignant potential were examined for possible TGF-beta(1)-mediated alterations in ODC expression. Selective induction of ODC gene expression occurred. This induction was dependent upon the cellular phenotype expressed and the mechanisms responsible for the regulation of the TGF-beta(1)-mediated alterations in ODC expression varied as a function of malignant potential. The TGF-beta(1)-mediated alterations in ODC gene expression involves de novo protein synthesis, transcriptional, and post-transcriptional events. Evidence is also 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 ODC expression in H-ras transformed cells. Evidence is also presented to suggest a possible role for cellular polyamines in the TGF-beta(1)-mediated alterations in ODC expression in H-ras transformed cells. Additionally, alterations in cellular polyamines were shown to influence TGF-beta(1) gene expression in H-ras transformed cells and that these alterations occurred, in part, through post-transcriptional events. The TGF-beta(1)-mediated regulation of ODC expression in H-ras transformed cells of varying degrees of malignant potential appears to be complex, multifaceted, and interactive. This study illustrates the importance of TGF-beta(1)-mediated regulation of ODC expression as a result of H-ras mediated cellular transformation and malignant progression, and further suggests that this TGF-beta(1)-mediated regulation constitutes an integral part of an altered growth regulatory program.

Insulin-mediated alterations in S-adenosylmethionine decarboxylase expression in H-ras transformed cells of varying degrees of malignancy.

Cell growth regulation is a highly complex process. The present study demonstrates a novel link between alterations in insulin-mediated regulation during malignant conversion and the expression of S-adenosylmethionine decarboxylase, a key regulatory activity in the biosynthesis of polyamines. H-ras transformed mouse 10 T 1/2 cell lines exhibiting increasing malignant potential were investigated for possible insulin-mediated changes in S-adenosylmethionine decarboxylase gene expression. Selective induction of S-adenosylmethionine decarboxylase gene expression was observed, because, in contrast to nontransformed 10T 1/2 cells, only H-ras transformed cells capable of only benign tumour formation or H-ras transformed metastatic cells exhibited marked elevations in S-adenosylmethionine decarboxylase mRNA levels. Evidence for regulation of S-adenosylmethionine decarboxylase gene expression at both transcriptional and post-transcriptional levels was found. Evidence was also found for a cycloheximide sensitive regulator of S-adenosylmethionine decarboxylase gene expression in H-ras transformed metastatic cells, whose effect, in combination with insulin, resulted in a further augmentation of S-adenosylmethionine decarboxylase gene expression. This regulation was not present in H-ras transformed cells capable of only benign tumour formation. These results suggest that insulin can modulate S-adenosylmethionine decarboxylase gene expression in H-ras transformed cells and further suggests a mechanism of insulin stimulation of transformed cells wherein alterations in the regulatory activity of S-adenosylmethionine decarboxylase gene expression are critical and constitutes a part of an altered growth regulatory program associated with cellular transformation.

Transforming growth factor beta1 selectively regulates ferritin gene expression in malignant H-ras-transformed fibrosarcoma cell lines.

Transforming growth factor beta1 is an important growth regulator in many cell types, usually exerting a negative effect on cellular growth. Inhibition of DNA synthesis and cell proliferation is frequently lost during malignant transformation, and in some cases, tumor cell proliferation is actually stimulated by TGF-beta1. The present study demonstrates a novel link between alterations in TGF-beta1 regulation during malignant conversion, and the expression of ferritin, an important activity involved in a number of biological functions including iron homeostasis and cell-growth control. A series of H-ras-transformed mouse 10 T 1/2 cell lines, exhibiting increasing malignant potential, was investigated for possible TGF-beta1-mediated changes in ferritin gene expression. Selective induction of gene expression was observed, since only H-ras-transformed cells with malignant potential exhibited marked elevations in ferritin gene expression, in particular, alterations in H-ferritin gene expression. The regulation of H-ferritin gene expression in response to TGF-beta1 did not involve alterations in transcription, but occurred through mechanisms of post-transcriptional stabilization of the H-ferritin mRNA. Additionally, evidence was obtained for a cycloheximide-sensitive regulator of H-ferritin gene expression, since the presence of this protein synthesis inhibitor increased H-ferritin message levels, and in combination with TGF-beta1, cooperated in an additive manner to augment H-ferritin gene expression. These results show for the first time that TGF-beta1 can regulate ferritin gene expression in malignant H-ras transformed cells, and suggest a mechanism for growth factor stimulation of malignant cells, in which early alterations in the control of H-ferritin gene expression are important.

Altered ornithine decarboxylase and S-adenosylmethionine decarboxylase expression and regulation in mouse fibroblasts transformed with oncogenes or constitutively active Mitogen-Activated Protein (MAP) kinase kinase.

In the present study, the expression and the regulation of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) was examined in a series of oncogene transformed cell lines. The expression of both ODC and SAMDC was found to correlate with the nature of the oncogene expressed and with the resulting cellular phenotype expressed. This study demonstrates, for the first time, that the expression of both ODC and SAMDC increases as a function of cellular transformation and, in particular, as a function of malignant progression. Ras transformed cells were shown to express a unique regulatory mechanism whereby a co-ordinate up-regulation of the expression of both ODC and SAMDC occurs (via post-transcriptional stabilization of their mRNA transcripts) in the presence of protein synthesis inhibition. Altered expression (and regulation) of both ODC and SAMDC is suggested to constitute an important part of an altered growth regulatory program inherent to oncogene transformed cells, in particular, to transformed cells capable of malignant progression.

Basic fibroblast growth factor selectively regulates ornithine decarboxylase gene expression in malignant H-ras transformed cells.

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.

Altered regulation of message stability and tumor promoter-responsive cis-trans interactions of ribonucleotide reductase R1 and R2 messenger RNAs in hydroxyurea-resistant cells.

Mammalian ribonucleotide reductase is a highly regulated activity essential for DNA synthesis and repair. The activity and message levels of the enzyme are elevated in cells treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, and this appears to be mediated through specific cis elements in the 3'-untranslated region of the R1 and R2 mRNAs that interact with R1 and R2 binding proteins called R1BP and R2BP, respectively. Hydroxyurea-resistant cells with increased R1 and R2 message levels were observed to have increased R1 and R2 message half-lives. This was accompanied by alterations in R1 and R2 3'-untranslated region cis-trans interactions, as judged by band shift and UV cross-linking assays, in which R1BP and R2BP binding was markedly reduced. This first description of mutant mammalian cells altered in message stability regulatory determinants indicates another mechanism for acquiring resistance to an antitumor agent. Furthermore, the present study strongly supports the concept that R1BP and R2BP are important general regulators of ribonucleotide reductase message stability and act as message destabilizing factors.

Increased phosphorylation of histone H1 in mouse fibroblasts transformed with oncogenes or constitutively active mitogen-activated protein kinase kinase.

We compared the nucleosomal organization, histone H1 subtypes, and histone H1 phosphorylated isoforms of ras-transformed and parental 10T1/2 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 10T1/2 cells had similar amounts of H1 subtypes, proteins that have a key role in the compaction of chromatin. However, labeling studies with 32P 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.

Malignant transformation by H-ras results in aberrant regulation of ribonucleotide reductase gene expression by transforming growth factor-beta 1.

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.

Ornithine decarboxylase gene expression is aberrantly regulated via the cAMP signal transduction pathway in malignant H-ras transformed cell lines.

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 of polyamines. Mouse 10T1/2 fibroblasts and a series of 10T1/2 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 10T1/2 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 cells with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) led to an elevation in ODC mRNA levels not observed in parental 10T1/2 fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in the cyclic AMP signal transduction pathway regulating ribonucleotide reductase gene expression in malignant H-ras transformed cell lines.

Ribonucleotide reductase is a highly regulated activity responsible for reducing ribonucleotides to deoxyribonucleotides, which are required for DNA synthesis and DNA repair. We have tested the hypothesis that malignant cell populations contain alterations in signal pathways important in controlling the expression of the two genes that code for ribonucleotide reductase, R1 and R2. A series of radiation and H-ras transformed mouse 10T1/2 cell lines with increasing malignant potential were exposed to stimulators of cAMP synthesis (forskolin and cholera toxin), an inhibitor of cAMP degradation (3-isobutyl-1-methylxanthine) and a biologically stable analogue of cAMP (8-bromo-cAMP). Dramatic elevations in the expression of the R1 and R2 genes at the message and protein levels were observed in malignant metastatic populations, which were not detected in the normal parental cell line or in cells capable of benign tumor formation. These changes in ribonucleotide reductase gene expression occurred without any detectable modifications in the rates of DNA synthesis, showing that they were regulated by a novel mechanism independent of the S phase of the cell cycle. Furthermore, studies with forskolin (a stimulator of the protein kinase A signal pathway) and the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (a stimulator of the protein kinase C signal pathway), alone or in combination, indicated that their effects on R1 and R2 gene expression in a highly malignant cell line were greater than when they were tested individually, suggesting that the two pathways modulating R1 and R2 gene expression can cooperate to regulate ribonucleotide reduction, and interestingly this can occur in a synergistic fashion. Also, a direct relationship between H-ras expression and ribonucleotide reductase gene expression was observed; analysis of forskolin mediated elevations in R1 and R2 message levels closely correlated with the levels of H-ras expression in the various cell lines. In total, these studies demonstrate that ribonucleotide reductase expression is controlled by a complex process, and malignant ras transformed cells contain alterations in the regulation of signal transduction pathways that lead to novel modifications in ribonucleotide reductase gene expression. This signal mechanism, which is aberrantly regulated in malignant cells, may be related to regulatory pathways involved in determining ribonucleotide reductase expression in a S phase independent manner during periods of DNA repair.

TGF-beta 1 stimulation of cell locomotion utilizes the hyaluronan receptor RHAMM and hyaluronan.

TGF-beta is a potent stimulator of motility in a variety of cell types. It has recently been shown that hyaluronan (HA) can directly promote locomotion of cells through interaction with the HA receptor RHAMM. We have investigated the role of RHAMM and HA in TGF-beta-stimulated locomotion and show that TGF-beta triggers the transcription, synthesis and membrane expression of the RHAMM receptor and the secretion of HA coincident with the induction of the locomotory response. This was demonstrated by both incubating cells with exogenous TGF-beta 1 and by stimulating the production of bioactive TGF-beta 1 in tumor cells transfected with TGF-beta 1 under the control of the metallothionein promoter. TGF-beta 1-induced locomotion was suppressed by antibodies that prevented HA/RHAMM interaction, using polyclonal antibodies to either RHAMM fusion protein or RHAMM peptides, or mAbs to purified RHAMM. Peptides corresponding to the HA-binding motif of RHAMM also suppressed TGF-beta 1-induced increases in motility rate. Spontaneous locomotion of fibrosarcoma cells was blocked by neutralizing secreted TGF-beta with panspecific TGF-beta antibodies and by inhibition of TGF-beta 1 secretion with antisense oligonucleotides. Polyclonal anti-RHAMM fusion protein antibodies and peptide from the RHAMM HA-binding motif also suppressed the spontaneous motility rate of fibrosarcoma cells. These data suggest that fibrosarcoma cell locomotion requires TGF-beta, and the pathway by which TGF-beta stimulates locomotion uses the HA receptor RHAMM and HA.

Transforming growth factor beta 1 selectively regulates ornithine decarboxylase gene expression in malignant H-ras transformed fibrosarcoma cell lines.

Negative growth regulators such as the transforming growth factor beta (TGF-beta) family appear to be important inhibitors in most tissue types. However, inhibition of DNA synthesis and cell proliferation is frequently lost during malignant transformation, and in some cases, tumor cell proliferation is actually stimulated by TGF-beta. The present study demonstrates a novel link between alterations in TGF-beta regulation during malignant conversion, and the expression of ornithine decarboxylase, a key rate-limiting activity in the biosynthesis of polyamines, and an enzyme that plays an important role in cell growth and differentiation. A panel of radiation and H-ras transformed mouse 10T1/2 cell lines exhibiting increasing malignant potential was investigated for possible TGF-beta 1 mediated changes in ornithine decarboxylase gene expression. Selective induction of gene expression was observed since only H-ras transformed cell lines with malignant potential exhibited marked elevations in ornithine decarboxylase message levels. Ornithine decarboxylase gene expression in nontransformed 10T1/2 cells and cell lines capable of only benign tumor formation was unaffected by TGF-beta 1 treatment. H-ras transformed cells were transfected with a plasmid placing the TGF-beta 1 coding region under the control of a zinc sensitive metallothionein promoter. When these cells were cultured in the presence of zinc an elevation of TGF-beta 1 mRNA was observed within 30 min. This increase in TGF-beta 1 message closely coincided with an elevation in ornithine decarboxylase message, and preceded an induction of jun-B, an early response gene in cells sensitive to TGF-beta 1 stimulation. Evidence for regulation of ornithine decarboxylase gene expression by TGF-beta 1 at both transcription and posttranscription was found. Actinomycin D pretreatment of malignant cells prior to TGF-beta 1 exposure prevented the increase in ornithine decarboxylase message. Marked differences in the rates of ornithine decarboxylase message decay were observed when cells treated with TGF-beta 1 were compared to untreated controls, with the half-life of ornithine decarboxylase mRNA increasing from 2.5 h in untreated cells to 17.5 h in cells exposed to TGF-beta 1. In addition, evidence was obtained for a cycloheximide sensitive regulator of ornithine decarboxylase gene expression, since the presence of this protein synthesis inhibitor increased the levels of ornithine decarboxylase message, and this effect was synergistically augmented by exposure of cells to cycloheximide and induction of TGF-beta 1 gene expression together.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta 1 mediated alterations in ribonucleotide reductase gene expression in BALB/c 3T3 fibroblasts.

Transforming growth factor-beta 1 (TGF-beta 1) stimulated DNA synthesis (3-fold) in BALBc/3T3 fibroblasts following 24 hours of growth factor exposure. Since ribonucleotide reductase is important for the coordination of DNA synthesis and cell proliferation, we investigated the hypothesis that cells like BALB/c 3T3, which are TGF-beta 1 responsive, would exhibit modifications in expression of the gene for ribonucleotide reductase following growth factor treatment. We observed 2.6, 4.1, and 4.8-fold increases in ribonucleotide reductase activity following TGF-beta 1 exposure for 6, 12, and 24 hours, respectively. Increased ribonucleotide reductase R2 gene expression (3, 3.7, and 4.5-fold) and R1 gene expression (2,2.5, and 2.6-fold) were observed following 6, 12, and 24 hours of TGF-beta 1 treatment, respectively. Western blots indicated 2.2, 3.1, and 4.1-fold increases in protein R2 levels at 6, 12, and 24 hours exposure to TGF-beta 1, whereas 2.6 and 3.3-fold elevations in R1 protein levels were observed at 12 and 24 hours post-TGF-beta 1 exposure. These TGF-beta 1 mediated modifications in ribonucleotide reductase gene expression occurred, in part, prior to any detectable changes in the rate of DNA synthesis, demonstrating alterations in the normal regulation of ribonucleotide reductase. Furthermore, these alterations could be markedly reduced by prolonged pretreatment with 12-O-tetradecanoylphorbol-13-acetate (R2 gene expression increased by only 1.3, 1.5 and 2.3-fold after 6, 12, and 24 hours of TGF-beta 1 treatment, respectively), suggesting a role for a protein kinase C pathway in the TGF-beta 1 regulated changes in ribonucleotide reductase gene expression. These results indicate for the first time that TGF-beta 1 can regulate the expression of the two genes for ribonucleotide reductase in BALB/c 3T3 fibroblasts, and suggest that regulation of these genes plays an important role in critical events involved in growth factor modulation of normal and transformed cell proliferation.