Immediate early-gene induction in rat osteoblastic cells after mechanical deformation.

Previous studies have reported changes in proliferation, second-messenger generation and activation of various cellular processes when osteoblasts have been mechanically stimulated. Recent evidence suggests that mechanical loading of long bones induces immediate early-gene expression. Immediate early genes, such as Egr-1, are genes that control cell proliferation, are involved in signal transduction, and share properties of transcription factors. The purpose of this study was to examine how mechanical deformation of osteoblasts affects cellular proliferation and Egr-1 mRNA induction. Osteoblasts were isolated from collagenase digestion of newborn rat calvariae, cultured in Petri dishes with flexible bottoms and then constantly stretched, producing an increase of 3 or 7% in surface area. A mechanical stretch of 7% for 0.5 or 24 h resulted in a doubling of [3H]thymidine incorporation, while 50 nM of epidermal growth factor resulted in a 4-fold increase. A time-course experiment showed that a 7% stretch induced Egr-1 mRNA as early as 15 mm, reaching maximum levels by 60 min and returning to baseline by 120 min. Epidermal growth factor at 50 nM for 60 min resulted in a 3.8-fold Egr-1 mRNA induction. A mechanical stretch of 3% for 30 min also produced an Egr-1 mRNA induction. No induction of Egr-1 mRNA was seen in osteoblasts that were exposed to conditioned media from deformed cells. It is concluded that the immediate early gene, Egr-1, may be directly involved in the signal-transduction pathway of mechanical stimuli in osteoblasts.

Differential regulation of p27 and cyclin D1 by TGF-beta and EGF in C3H 10T1/2 mouse fibroblasts.

Previously, we found that stimulation of C3H 10T1/2 mouse fibroblasts with TGF-beta leads to the striking and rapid down-regulation of p27kip1 expression during G1 phase. Here, we demonstrate that TGF-beta treatment of C3H 10T1/2 cells does not alter the steady-state level of Kip1 message sufficiently to account for the observed down-regulation of p27. This demonstrates that TGF-beta-induced down regulation of p27kip1 occurs at a post-transcriptional level, consistent with a degradative mechanism of p27kip1 down-regulation. Epidermal growth factor (EGF) does not lead to the rapid down-regulation of p27 observed following treatment of cells with TGF-beta. Also in contrast with TGF-beta, EGF causes a strong upregulation of cyclin D1, while neither growth factor affects cdk4 protein levels. These results imply that in this cell type TGF-beta overcomes an inhibitory threshold to cdk activation by cyclin-dependent kinase inhibitors primarily through down-regulation of p27, while EGF overcomes this threshold predominantly through upregulation of cyclin D1 levels. This divergence in pathways may explain why TGF-beta-induced cell cycle kinetics are slower than those of EGF in these cells, and the ability of TGF-beta to delay EGF-induced cell cycle kinetics to its own, slower kinetics. In support of this hypothesis, TGF-beta prevents EGF-induced upregulation of cyclin D1 levels, while TGF-beta is still able to induce p27 down-regulation even in the presence of EGF. In contrast to the case with p27 degredation, neither TGF-beta nor EGF have an observable effect on the steady-state levels of p21 in this cell type.

Triplex forming ability of a c-myc promoter element predicts promoter strength.

Most transcription factors are believed to bind promoter elements in the B-DNA conformation, wherein linear sequence determines specificity. However, there are promoter elements that can form complex structures such as intramolecular triplexes, and these structures may participate in the activity of these promoter elements. We have previously shown that a c-myc promoter element, termed the nuclease-sensitive element or NSE, can form tandem intramolecular triplexes of the H-DNA type and has a repeating sequence motif (ACCCTCCCC)4. The NSE was mutated and examined for transcriptional activity and for intra- and intermolecular triplex forming ability. The transcriptional activity of mutant NSEs can be predicted by the element's ability to form H-DNA and not by repeat number, position, or the number of mutant base pairs. DNA may therefore be a dynamic participant in the transcription of the c-myc gene.

Transforming growth factor beta 1-induced delay of cell cycle progression and its association with growth-related gene expression in mouse fibroblasts.

TGF beta-induced cell cycle progression is relatively slower than that induced by EGF or PDGF-BB. Further, TGF beta delays EGF or PDGF-induced 5-phase entry in C3H 10T1/2 mouse fibroblasts. In accordance with this delay, the induction of mRNA level of 'immediate early genes' such as c-myc, c-fos, c-jun and junB by TGF beta has slower kinetics compared with those of EGF. TGF beta induces c-sis gene, suggesting possible involvement of secondary growth stimulation by PDGF-like proteins. However, anti-PDGF-AB antibody, which was inhibitory to FDGF-BB-induced [3H]thymidine incorporation, did not block TGF beta-induced DNA synthesis. These results first demonstrate that the delay of cell cycle progression by TGF beta is closely associated with the altered regulation of growth-related gene expression in fibroblasts.

The identification of a tandem H-DNA structure in the c-myc nuclease sensitive promoter element.

Previous studies have shown that the c-myc nuclease sensitive element (NSE) is capable of forming H-DNA in vitro. The NSE sequence exhibits strong purine/pyrimidine strand asymmetry. To study the NSE further, we have isolated the element from other c-myc sequences and have shown that the NSE alone is sufficient for the formation of H-DNA in supercoiled plasmids. We also show that the NSE forms a complex structure containing both H-y3 and H-y5 H-DNA. We term this structure tandem H-DNA.

Regulation of C-myc protooncogene expression in osteoblastic cells by arachidonic acid metabolites: relationship to proliferation.

Prostaglandin E2 and leukotriene B4 are metabolites of arachidonic acid with well-characterized effects on osteoblastic cells. Prostaglandin E2 has been shown to be a potent bone-resorbing agent and to stimulate as well as inhibit osteoblastic cell proliferation. Leukotriene B4 has also been demonstrated to stimulate or inhibit osteoblastic cell proliferation, depending on the cell type tested. In the present study, the potential relationship of the effects of prostaglandin E2 and leukotriene B4 on osteoblastic cell proliferation to c-myc protooncogene expression was investigated. Prostaglandin E2 has been shown previously to inhibit normal rat osteoblastic cell proliferation. The present studies show that prostaglandin E2 at 10(-6) M decreased c-myc expression in these cells. In the human osteoblastic osteosarcoma cell line, G292, prostaglandin E2 increased c-myc expression and inhibited proliferation. In contrast, epidermal growth factor increased DNA synthesis as well as c-myc expression. Prostaglandin E2 also inhibited proliferation of another human osteoblastic osteosarcoma cell line, Saos-2, but it did not produce any changes in c-myc expression. In these cells, epidermal growth factor did not affect either DNA synthesis or c-myc expression. Leukotriene B4 did not show any effects on c-myc expression in any of the osteoblastic cells tested.

Defective c-myc and c-myb RNA turnover in acute myeloid leukemia cells.

Dysregulated expression of the c-myc and c-myb protooncogenes has been implicated in the pathogenesis of acute myeloid leukemia (AML). To elucidate mechanisms of c-myc dysregulation in AML cells, we studied c-myc RNA turnover in peripheral blood blasts from eight patients using actinomycin D transcription blockade. Rapid c-myc RNA turnover was seen in cells from six patients, with half-lives of approximately 30 minutes, similar to those reported in normal myeloid cells, in HL-60 cells, and in other cell lines. c-myc RNA turnover was prolonged in cells of the other two patients, with half-lives of greater than 75 minutes. c-fos RNA turnover was rapid in blasts from all eight patients, with half-lives of approximately 15 minutes. Stabilization of GM-CSF transcripts was not observed. In contrast, c-myb RNA half-lives were greater than 75 minutes in cells of the two patients with prolonged c-myc RNA turnover, as compared to 30 minutes in cells of the other six patients. Enhanced stability of both c-myc and c-myb RNA species suggests that a defect exists in a trans-acting factor that destabilizes both of these normally labile RNAs. Incomplete correlation between c-myc RNA levels and half-lives indicates regulation of c-myc expression at the level of transcription or nuclear transport in addition to posttranscriptional regulation.

A CT promoter element binding protein: definition of a double-strand and a novel single-strand DNA binding motif.

Numerous genes contain promoter elements that are nuclease hypersensitive. These elements frequently possess polypurine/polypyrimidine stretches and are usually associated with altered chromatin structure. We have previously isolated a clone that binds a class of CT-rich promoter elements. We have further characterized this clone, termed the nuclease-sensitive element protein-1, or NSEP-1. NSEP-1 binds both duplex CT elements and the CT-rich strand of these elements in a 'generic' sequence specific manner and has overlapping but distinct single-and double-strand DNA binding domains. The minimal peptide region sufficient for both duplex and single-strand DNA binding includes two regions rich in basic amino acids flanking an RNP-CS-1 like octapeptide motif. Deletion analysis shows that the single-strand DNA binding activity is mediated by the RNP-CS-1 like octapeptide motif and is the key peptide region necessary for single-strand binding. NSEP-1's affinity for CT rich promoter elements with strand asymmetry in addition to its double- and single-strand DNA binding properties suggests that it may be a member of a class of DNA binding proteins that modulate gene expression by their ability to recognize DNA with unusual secondary structure.

Ribonucleoprotein and protein factors bind to an H-DNA-forming c-myc DNA element: possible regulators of the c-myc gene.

We have located a positive, cis-acting DNA sequence element within the 5' flanking DNA of the c-myc gene (-125 base pairs). This DNA sequence element has a large purine-pyrimidine strand asymmetry and can assume the H-DNA conformation. A factor with the properties of a ribonucleoprotein (RNP) interacts with this DNA region. The interaction of the c-myc DNA sequence element and the RNP involves an RNase H-sensitive mechanism and, therefore, may involve an RNA.DNA hybrid. In addition, a protein factor(s) binds to this DNA sequence element. DNA footprinting and mutant oligonucleotide binding/competition assays implicate a punctate, poly(G.C) recognition/binding sequence for the RNP factor, whereas the major protein factor requires two ACCCT sequence motifs for maximal binding. These results suggest that RNP and protein factors act as positive transcriptional regulators of the c-myc gene, perhaps by altering DNA topology.

Induction of c-fos and c-myc proto-oncogene expression by epidermal growth factor and transforming growth factor alpha is calcium-independent.

Intracellular calcium has been proposed to be an important mediator of signal transduction by various growth factors. We have studied the role of intracellular calcium in the mitogenic stimulation of C3H 10T1/2 mouse fibroblasts by epidermal growth factor and transforming growth factor alpha. We have found that both these peptides can cause a marked, transient increase in intracellular calcium levels. This rise occurs only in the presence of extracellular calcium. However, this calcium transient is not involved in the accumulation of c-fos and c-myc mRNAs which are elicited by these growth factors, since mRNA induction is observed to an equivalent degree in the absence or presence of extracellular calcium. These results demonstrate that although these growth factors cause an increase in intracellular calcium, the calcium second messenger system is not responsible for the induction of c-fos and c-myc mRNAs in C3H 10T1/2 fibroblasts.

cis-acting determinants of basal and lipid-regulated apolipoprotein A-IV expression in mice.

The levels of apolipoprotein A-IV (apoA-IV) mRNA are regulated by dietary lipid in the liver of both the mouse and rat. Thirteen different inbred mouse strains were fed a high lipid diet, and the effect on apoA-IV liver mRNA levels was examined. It was found that each strain responded in one of two ways. Mice of four strains had higher liver apoA-IV mRNA levels as compared with syngeneic mice fed a normal chow diet. Mice of the other nine strains had decreased liver apoA-IV mRNA levels as compared with syngeneic mice fed a normal chow diet. Using F1 hybrids between mice from BALB/c, C3H, and C57BL/6 and between 129 and C57BL/6, as well as recombinant inbred strains derived from a cross between BALB/c and C57BL/6, we have shown that both the normal level of liver apoA-IV mRNA in the chow-fed mice and the lipid-dependent regulation of apoA-IV mRNA levels are controlled by cis-acting genetic elements. The apoA-IV mRNA levels in mice fed a normal diet varied dramatically among strains, with the largest difference (90-fold) being between the 129/J inbred strain and the C57BL/6J strain. In addition, we have examined the expression of apoA-IV during mouse development. ApoA-IV mRNA is expressed early in mouse liver (16 days postcoitum), whereas others have shown previously that rat liver apoA-IV mRNA is undetectable until 14 days after birth. ApoA-IV mRNA levels in the intestine and apoA-I mRNA levels in the liver and intestine, by contrast, mirror the pattern seen in the rat.

A cis-acting transcription element of the c-myc gene can assume an H-DNA conformation.

I have used chemical probes and an oligonucleotide-association assay to determine the structure of a nuclease-sensitive, c-myc DNA region. I find that this DNA region can form a triplex-single stranded conformer in vitro--the H-DNA conformer. This DNA region has been shown previously to be a positive, cis-acting transcription element of the c-myc gene and to bind nuclear factors, including a base-paired ribonucleoprotein. Therefore, H-DNA may be a functionally important in vivo topoisomer where the H-DNA and B-DNA conformers have different transcriptional activities.

T-cell receptor gene rearrangement and its expression in human myeloid leukemia cell lines.

ML cell lines (ML-1, -2, and -3) were derived from the cells of a patient with acute myelocytic leukemia preceded by a T-cell malignant lymphoma. A deletion of chromosome 11 (11q-) was common to the affected cells in both neoplastic phases. We report here that the three ML cell lines have DNA rearrangements of the T-cell receptor (TcR)-beta and gamma-chain genes in addition to immunoglobulin heavy-chain gene rearrangement, though they do not have TcR gene messages. The findings presented here indicate that ML cell lines could be used as models for the elucidation of the bilineal nature of hematopoietic neoplastic cells, though they have a biphenotypic (myelomonocytic/T-cell) marker expression.

c-myc RNA degradation in growing and differentiating cells: possible alternate pathways.

Transcripts of the proto-oncogene c-myc are composed of a rapidly degraded polyadenylated RNA species and an apparently much more stable, nonadenylated RNA species. In this report, the extended kinetics of c-myc RNA turnover have been examined in rapidly growing cells and in cells induced to differentiate. When transcription was blocked with actinomycin D in rapidly growing cells, poly(A)+ c-myc was rapidly degraded (t1/2 = 12 min). c-myc RNA lacking poly(A) initially remained at or near control levels; however, after 80 to 90 min it was degraded with kinetics similar to those of poly(A)+ c-myc RNA. These bizarre kinetics are due to the deadenylation of poly(A)+ c-myc RNA to form poly(A)- c-myc, thereby initially maintaining the poly(A)- c-myc RNA pool when transcription is blocked. In contrast to growing cells, cells induced to differentiate degraded both poly(A)+ and poly(A)- c-myc RNA rapidly. The rapid disappearance of both RNA species in differentiating cells suggests that a large proportion of the poly(A)+ c-myc RNA was directly degraded without first being converted to poly(A)- c-myc RNA. Others have shown that transcriptional elongation of the c-myc gene is rapidly blocked in differentiating cells. We therefore hypothesize that in differentiating cells a direct, rapid degradation of poly(A)+ c-myc RNA may act as a backup or fail-safe system to ensure that c-myc protein is not synthesized. This tandem system of c-myc turnoff may also make cells more refractory to mutations which activate constitutive c-myc expression.

Abnormally banded region in a poorly differentiated sarcoma is not correlated with amplification of c-MYC or c-MOS protooncogenes.

A poorly differentiated sarcoma in a 32-year-old female revealed a large, abnormally banded region in one chromosome #8 in all metaphases. The modal karyotype was 46,XX, -8, +mar. Southern blot hybridization was performed with probes for two protooncogenes located in chromosome 8q (c-MYC and c-MOS). No amplification or rearrangement was observed to account for the cytogenetic abnormality.

myb oncogene in human hematopoietic neoplasia with 6q- anomaly.

Molecular and cytogenetic analyses were performed on human T-cell leukemia cell lines (PEER and MOLT-4) with the 6q- anomaly. The PEER cells contained an interstitial deletion of the long arm of chromosome 6, that is, del(6)(q13q21), as well as other changes. The MOLT-4 cells showed a terminal deletion of the long arm of chromosome 6, that is, del(6)(q24). The 700-bp BamHI/XbaI-digested c-myb probe hybridized to a 4.3-kb fragment in EcoRI digested DNAs from these two cell lines, showing no deletion, rearrangement, or amplification. On the other hand, ML cells [ML-1, -2 and -3; human myeloid/T-cell biphenotypic leukemia cell lines with del(6)(q24)] showed an amplification of the c-myb gene and had a high level of the c-myb-related mRNA at 3.5 kb. Though no amplification of the c-myb at the DNA level was noted in the PEER or MOLT-4 cell lines, apparent high expression of the c-myb was detected in these human T-cell neoplastic lines. These results indicate that high c-myb expression is related to lineage of hematopoietic neoplasia rather than to the 6q- change.

Transforming growth factor alpha (TGF alpha) induction of C-FOS and C-MYC expression in C3H 10T1/2 cells.

We have investigated the effects of transforming growth factor alpha (TGF alpha) in C3H10T1/2 cells, on S phase entry and early gene activation events associated with cell cycle progression. We find that EGF and TGF alpha, which both utilize the EGF receptor for signal generation, are able to stimulate DNA synthesis in these cells with nearly superimposable kinetics; however, the stimulation by TGF alpha was slightly greater at nearly all time points assayed. This report is the first showing that TGF alpha, like EGF, vigorously induces c-myc and c-fos gene expression in these cells. A significant stimulation of c-myc and c-fos mRNA levels is observed with both TGF alpha and EGF; c-myc mRNA levels show an 8-fold induction with both mitogens, while c-fos inductions were on the order of 12 to 14-fold at maximum. However, the induction of c-myc mRNA by TGF alpha has slower kinetics than by EGF.

Hu-ets-1 gene in congenital leukemia with t(11;19)(q23;p13).

Cytogenetic analysis of the leukemic cells from a 1-day-old baby with an acute myelomonocytic leukemia revealed them to contain a chromosome change of t(11;19)(q23;p13). Molecular studies using a 980 bp HindIII/HpaI digested v-ets probe showed no DNA rearrangements, deletions, or amplification in the leukemic cells, including the JH immunoglobulin and T-cell receptor (alpha or beta) genes. The findings indicate that the leukemic cells with t(11;19)(q23;p13) appear not to contain a transposition or rearrangement of the protooncogene Hu-ets-1 located at 11q23, as previously described in leukemic cells with t(4;11)(q21;q23) and t(9;11)(p22;q23). The leukemic cases with t(11;19)(q23;p13) studied by us showed a phenotype compatible with their myelomonocytic nature, although it is possible that other cases may have a lymphoid phenotype.