Platelet-derived growth factor-stimulated c-myc RNA accumulation in MG-63 human osteosarcoma cells is independent of both protein kinase A and protein kinase C.

Treatment of quiescent MG-63 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) or platelet-derived growth factor (PDGF) stimulates the rapid accumulation of c-myc RNA. We have now determined that a similar effect can be induced by cAMP. Treatment with forskolin (an activator of adenylate cyclase), IBMX (a phosphodiesterase inhibitor), PGE1, and isoproterenol stimulated accumulation of both cAMP and c-myc RNA, but no increase in either cAMP or c-myc RNA was seen with the inactive forskolin analog 1,9-dideoxyforskolin. Forskolin and IBMX acted synergistically in stimulating accumulation of both cAMP and c-myc RNA. However, three lines of evidence indicated that PDGF action is not mediated by cAMP. First, PDGF treatment caused no elevation of cAMP within 1 h, even in the presence of IBMX. Second, the kinetics of c-myc RNA elevation after treatment with PDGF or forskolin were similar, ruling out delayed onset of cAMP stimulation. Finally, simultaneous treatment with forskolin and the calcium ionophore A23187 enhanced the elevation of c-myc RNA levels; no such effect was seen with PDGF. We had previously shown that PDGF action is not affected by prior treatment of MG-63 cells with TPA, a treatment which desensitizes the c-myc response to TPA. Similarly, TPA pretreatment had minimal effect on forskolin or IBMX-induced c-myc expression. These data suggest that cAMP, phorbol esters, and PDGF act independently to stimulate c-myc RNA expression in MG-63 cells. However, nuclear runoff experiments and RNA half-life measurements demonstrated that PDGF, phorbol ester, and cAMP all act to increase the transcription of the MYC gene.

Regulation of the transcript for a lysosomal protein: evidence for a gene program modified by platelet-derived growth factor.

Platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize MEP, a lysosomal protein. This enhanced synthesis appears to be largely regulated by the PDGF-modulated accumulation of MEP mRNA, a 1.8-kilobase species. The increase in the MEP transcript, which is dependent on the PDGF concentration, begins 3 to 4 h after PDGF addition and is maximal at 12 h. The accumulation of the MEP transcript is growth-factor specific: PDGF and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, an agent which acts like PDGF, induce MEP RNA accumulation, whereas epidermal growth factor, somatomedin C, insulin, and whole plasma do not. A spontaneously transformed BALB/c-3T3 cell line (ST2-3T3), which does not require PDGF for growth, optimally expresses MEP RNA in the absence of PDGF. The PDGF-modulated increase in MEP RNA is unlike PDGF-modulated c-myc and c-fos RNA accumulation because it is blocked by cycloheximide, suggesting a requirement for de novo protein synthesis. It appears that PDGF modulates a program of gene expression with the accumulation of some transcripts, typified by MEP, being dependent upon the translation of others.

Inhibition of N-myc expression and induction of apoptosis by iron chelation in human neuroblastoma cells.

Neuroblastoma is the second most common solid malignancy of childhood. Enhanced expression of the amplified N-myc gene in the tumor cells may be associated with poor patient prognosis and may contribute to tumor development and progression. The use of deferoxamine mesylate (DFO), an iron chelator, to treat neuroblastoma is being investigated in national clinical studies. We show here by TUNEL assay and DNA laddering that DFO induces apoptosis in cultured human neuroblastoma cells, which is preceded by a decrease in the expression of N-myc and the altered expression of some other oncogenes (up-regulating c-fos and down-regulating c-myb) but not housekeeping genes. The decrease in N-myc expression is iron-specific but does not result from inhibition of ribonucleotide reductase, because specific inhibition of this iron-containing enzyme by hydroxyurea does not affect N-myc protein levels. Nuclear run-on and transient reporter gene expression experiments show that the decrease in N-myc expression occurs at the level of initiation of transcription and by inhibiting N-myc promoter activity. Comparison across neuroblastoma cell lines of the amount of residual cellular N-myc protein with the extent of apoptosis measured as pan-caspase activity after 48 h of iron chelation reveals no correlation, suggesting that the decrease in N-myc expression is unlikely to mediate apoptosis. In conclusion, chelation of cellular iron by DFO may alter the expression of multiple genes affecting the malignant phenotype by multiple pathways. Given the clinical importance of N-myc overexpression in neuroblastoma malignancy, decreasing N-myc expression by DFO might be useful as an adjunct to current

Insulin-like growth factor-I induces cyclin-D1 expression in MG63 human osteosarcoma cells in vitro.

The insulin-like growth factors (IGFs) stimulate cell division by modulating events occurring during the prereplicative (G1) phase of the cell cycle, but identification of the critical events has proved difficult. Recent observations suggest that progression through the cell cycle is dependent on the activation of a group of serine-threonine-specific protein kinases whose activities are regulated by accessory proteins, termed cyclins. The identification of cyclin species expressed during G1 has led to the hypothesis that modulation of cyclin expression may be the critical event regulated by growth factors. The present studies were undertaken to determine whether the IGFs regulate the expression of specific G1 cyclins in MG63, a human cell line that is unusually responsive to IGF, and to characterize this effect. We found that in these cells IGF-I stimulates the cyclin-dependent kinases, and that stimulation is associated with an increase in cyclin-D1 mRNA and protein expression. The increase in cyclin-D1 occurs early in G1 and corresponds to the portion of the cell cycle in which IGF acts on these cells. The increase in cyclin-D1 mRNA is due at least in part to an increase in the rate of transcription initiation of the gene. The mRNA levels of cyclin-B1 (a G2 cyclin) and two cyclin-dependent kinases, cdc2 and cdk2, also increased in response to IGF, but at later times. These results are consistent with the hypothesis that IGF modulation of D-type cyclin expression plays a role in the regulation of cell replication.

Steady-state mRNA expression for growth factors in DMBA-induced rat mammary tumors.

Steady-state mRNA levels were examined for insulin-like growth factors (IGF) I and II, transforming growth factor alpha (TGF alpha) and its receptor and the epidermal growth factor (EGF) receptor in mammary tumors induced by DMBA in rats. An abundant 4.8 kb TGF alpha transcript was identified in all tumors, along with a 7.5-8.0 kb IGF-I transcript. A presumptive 2.9 kb IGF-II transcript was also identified in all tumors. Northern analyses and receptor autophosphorylation studies failed to detect EGF receptors in any mammary tumors. These findings suggest the potential for autocrine or paracrine influences of IGF-I and IGF-II in this tumor model and a possible paracrine influence of TGF alpha in tumor-induced neovascularization.

Chronic metabolic acidosis reversibly inhibits extracellular matrix gene expression in mouse osteoblasts.

Chronic metabolic acidosis induces net calcium efflux from bone mineral through an increase in osteoclastic resorption and a decrease in osteoblastic matrix deposition and mineralization. To determine the effects of chronic metabolic acidosis on the expression of genes necessary for mineralization, we grew primary bone cells, which are principally osteoblasts, to confluence in neutral pH (7.5) medium and then switched the cells either to a neutral pH or to an acidic pH (7.1) differentiation medium. Cells were harvested for RNA at 4- to 7-day intervals for up to 44 days. By 36 days, there was extensive bone nodule formation and mineralization in cells cultured in neutral medium; however, there was a substantial decrease in nodule formation and mineralization in cells cultured in acidic medium. There was a marked increase in matrix Gla protein RNA and an increase in osteopontin RNA in neutral cultures; however, acidic medium almost completely prevented any increase. In contrast, RNA levels for osteonectin and transforming growth factor-beta1 were not altered by chronic acidosis. Additional cells were incubated in acid differentiation medium for 1, 2, or 3 wk and then transferred to neutral medium; in each case, there was recovery of matrix Gla protein RNA and osteopontin RNA expression. Still other cells were incubated in neutral differentiation medium for 1, 2, or 3 wk and then transferred to acid medium; in each case there was inhibition of matrix Gla protein RNA and osteopontin RNA expression. Thus metabolic acidosis appears to specifically inhibit RNA accumulation of certain genes whose products may be essential for formation of mature bone matrix.

The effects of acid on bone.

Metabolic acidosis induces calcium efflux from bone and in the process buffers the additional hydrogen ions. Initially metabolic acidosis stimulates physicochemical mineral dissolution and then cell-mediated bone resorption. Acidosis increases activity of the bone resorbing cells, the osteoclasts, and decreases activity of the bone forming cells, the osteoblasts. Osteoblastic immediate early response genes are inhibited as are genes controlling matrix formation.

In vitro metabolic and respiratory acidosis selectively inhibit osteoblastic matrix gene expression.

Clinically, a decrease in blood pH may be due to either a reduction in bicarbonate concentration ([HCO(-)(3)], metabolic acidosis) or an increase in PCO(2) (respiratory acidosis). In mammals, metabolic acidosis induces a far greater increase in urine calcium excretion than respiratory acidosis. In cultured bone, metabolic acidosis induces a marked increase in calcium efflux and a decrease in osteoblastic collagen synthesis, whereas isohydric respiratory acidosis has little effect on either parameter. We have shown that metabolic acidosis prevents the normal developmental increase in the expression of RNA for matrix Gla protein and osteopontin in chronic cultures of primary murine calvarial bone cells (predominantly osteoblasts) but does not alter expression of osteonectin. To compare the effects of isohydric metabolic and respiratory acidosis on expression of these genes, bone cell cultures were incubated in medium at pH approximately 7.2 to model metabolic ([HCO(-)(3)], approximately 13 mM) or respiratory (PCO(2), approximately 80 mmHg) acidosis or at pH approximately 7.4 as a control. Cells were sampled at weeks 4, 5, and 6 to assess specific RNA content. At all time periods studied, both metabolic and respiratory acidosis inhibited the expression of RNA for matrix Gla protein and osteopontin to a similar extent, whereas there was no change in osteonectin expression. In contrast to the significant difference in the effects of metabolic and respiratory acidosis on bone calcium efflux and osteoblastic collagen synthesis, these two forms of acidosis have a similar effect on osteoblastic RNA expression of both matrix Gla protein and osteopontin. Thus, although several aspects of bone cell function are dependent on the type of acidosis, expression of these two matrix genes appears to be regulated by extracellular pH, independently of the type of acidosis.

Acute metabolic acidosis inhibits the induction of osteoblastic egr-1 and type 1 collagen.

Metabolic acidosis induces net calcium efflux from bone through a decrease in osteoblastic formation and an increase in osteoclastic resorption. We tested the hypothesis that changes in external pH would alter the expression of genes critical to the function of mouse calvarial bone cells, predominantly osteoblasts. Cells were cultured in physiologically neutral pH medium until confluent and then stimulated with fresh medium at either neutral or acidic pH. Among a group of immediate early response genes, including egr-1, junB, c-jun, junD, and c-fos, only egr-1 stimulation was modulated by changes in medium pH. At pH 7.4, RNA for egr-1 was stimulated approximately 10- to 30-fold, 40 min after medium change. A progressive decrease in pH to 6.8 led to a parallel reduction in egr-1 stimulation, and an increase in pH to 7.6 led to an increase in egr-1 stimulation. The protein synthesis inhibitor cycloheximide led to a superinduction of egr-1 with preservation of the pH dependency of expression. Osteoblasts synthesize collagen, which is subsequently mineralized. RNA for type 1 collagen was stimulated approximately three- to fivefold, 40 min after medium change. Again the stimulation was inhibited by acidosis and increased by alkalosis. Cycloheximide abolished the pH dependency of expression. These results suggest that small changes in external pH have a significant effect on the expression of certain genes important for osteoblastic function.

Platelet-derived growth factor-induced c-myc RNA expression. Analysis of an inducible pathway independent of protein kinase C.

Platelet-derived growth factor (PDGF) is generally considered to stimulate phosphoinositide turnover resulting in activation of protein kinase C and increased cytoplasmic [Ca2+]. We have examined the role of these secondary effects in regulation of c-myc mRNA accumulation in the MG-63 human osteogenic sarcoma line. Treatment of quiescent cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA) to down-regulate protein kinase C inhibited TPA-stimulated c-myc expression but did not affect the PDGF-modulated process. When cytoplasmic [Ca2+] was increased by addition of a Ca2+ ionophore (A23187 or ionomycin), no stimulation of c-myc RNA was seen; furthermore, these agents did not enhance the PDGF-modulated c-myc expression. Addition of EGTA to cultures treated with both PDGF and a Ca2+ ionophore did not inhibit c-myc induction but rather caused a superinduction of c-myc RNA accumulation. Superinduction occurred only if the [EGTA] was greater than [Ca2+] in the medium. This superinduction was distinct from the increased induction caused by inhibition of protein synthesis. Because PDGF-induced c-myc expression is independent of protein kinase C and increased cytoplasmic [Ca2+], the evidence suggests that PDGF modulates c-myc RNA accumulation in MG-63 cells via a novel pathway, seemingly uncoupled from the classic action of increased phosphoinositide metabolism.

Cloning of immunity and structural genes for colicin V.

The colicin V immunity and structural genes of plasmid pColV-B188 were cloned into the vectors pMB9, pBR322, and pMK16. Both genes are closely linked and can be isolated on a 900-base-pair deoxyribonucleic acid fragment. Insertion of the transposon Tn5 into this cloned sequence led to the construction of a mutant plasmid which conferred colicin V immunity, but not the ability to produce this colicin. Analysis of the products determined by these cloned genes in cells has led to the conclusion that the polypeptide involved in immunity has a molecular weight of about 6,500, whereas the colicin has a molecular weight of approximately 4,000.