Alan J. Kinniburgh

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

Previously, we found that stimulation of C3H 10T½ mouse fibroblasts with TGF‐β leads to the striking and rapid down‐regulation of p27Kip1 expression during G1 phase. Here, we demonstrate that TGF‐β treatment of C3H 10T½ 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‐β‐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‐β. Also in contrast with TGF‐β, 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‐β 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‐β‐induced cell cycle kinetics are slower than those of EGF in these cells, and the ability of TGF‐β to delay EGF‐induced cell cycle kinetics to its own, slower kinetics. In support of this hypothesis, TGF‐β prevents EGF‐induced upregulation of cyclin D1 levels, while TGF‐β is still able to induce p27 down‐regulation even in the presence of EGF. In contrast to the case with p27 degradation, neither TGF‐β nor EGF have an observable effect on the steady‐state levels of p21 in this cell type.

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.

Transforming growth factor alpha (TGFα) induction of c-fos and c-myc expression in C3H 10Tl/2 cells

Summary We have investigated the effects of transforming growth factor α (TGFα) in C3H10Tl/2 cells, on S phase entry and early gene activation events associated with cell cycle progression. We find that EGF and TGFα, 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 TGFα was slightly greater at nearly all time points assayed. This report is the first showing that TGFα, 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 TGFα 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 TGFα has slower kinetics than by EGF.

Differing patterns of proto-oncogene expression in immature and mature myeloid cells

Comparisons of the level of proto-oncogene expression in neoplastic cells and in normal cells are being made to determine the role of these genes in neoplastic development. Recent papers have reported that leukemic cells differ from normal cells in having higher c-myc RNA levels. One problem in interpreting these data is that leukemic and normal cell populations differ in the proportion of immature cells present in each. The studies described here, using chronic phase chronic myelogenous leukemia (CML) cells, compared the level of proto-oncogene expression in immature and mature myeloid cells. Substantial differences in the level and pattern of expression were found with the immature cells containing higher c-myc RNA levels and the mature cells containing higher histone H3 RNA levels. c-fos RNA levels parallel the distribution of monocytes. While the c-myc RNA level in the CML cell population as a whole is similar to that in normal marrow cell populations and less than that in the bone marrow cells of patients with acute myelogenous leukemia (AML), c-myc RNA levels in subpopulations of immature chronic phase CML myeloid cells approximate that found in AML cells. Additionally, the studies described here suggest that the presence of high c-myc and c-fos RNA levels in light density immature cells may be a unique characteristic of acute myeloid leukemic cells.

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.

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.

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.

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.

Breakpoint cluster region rearrangements in chronic myelogenous leukemia with a masked Philadelphia chromosome

Cytogenetic and molecular analyses were performed in a case of chronic myelogenous leukemia. The cytogenetic study revealed that the leukemic cells of this patient contained a three-way translocation involving chromosomes #5, #9, and #22, resulting in a masked Philadelphia chromosome; the karyotype of the leukemic cells was 46,XY,t(5;22;9)(q31;q11;q34). Southern blot analysis of leukemic cell DNA was performed using a 1.1 kb HindIII-EcoRI breakpoint cluster region (bcr) probe. The results showed that BglII digested DNA showed two abnormal bcr fragments (i.e., 5.2 kb and 2.7 kb) in contrast to the results with DNA from two patients with a standard Ph chromosome [t(9;22)(q34;q11)] who showed one normal 5.0-kb bcr fragment in addition to altered fragments of about 4.3 kb or 4.0 kb. Bam HI digests of DNA from the leukemic cells of the patient with the masked Ph chromosome showed two bands (3.3 kb and 6.5 kb), whereas, DNA from the two patients with standard Ph translocations showed only a 3.3-kb bcr fragment. The results indicate that the molecular events in a masked Ph affect the bcr locus in a manner similar to that seen in standard Ph chromosomes.

Human-fms gene is retained in acute lymphoblastic leukemia cells with del(5)(q32)

Cytogenetic and molecular investigations of NALM 6 cells (a pre-B-lymphoblastic acute leukemia cell line) revealed them to contain both alleles of the c-fms gene, though the cells had chromosomal changes of 5q- and 12p+. The amount of DNA fragments hybridized to the 1.4 kb PstI/PstI v-fms probe in the NALM 6 cells was approximately the same, when compared with cells of an Epstein-Barr virus-transformed lymphoblastoid cell line with a normal karyotype. Chromosome banding analysis revealed that the breakpoint of the 5q- in the NALM 6 cells was at the proximal portion of the 5q32 band. Chromosomal in situ hybridization of NALM 6 cells showed a significant accumulation of grains on the terminal portions of the abnormal 5q- chromosomes (5q32), as well as on the normal chromosomes #5 with a peak at 5q32-q33. These findings indicate that the human c-fms gene is not deleted in the lymphoblastic leukemia cells with a 5q- studied by us and that it does not show rearrangement or amplification. Thus, the results indicate that a difference in the dosage of the c-fms gene in acute lymphoblastic leukemia cells with the 5q- versus that in cells with the 5q- change in nonlymphocytic neoplasia; in the latter a hemizgosity of the c-fms gene has been suggested.