Expression of c-myc proto-oncogene during podophyllotoxin induced IW32 erythroleukemia cell differentiation

The effect of hemin or podophyllotoxin on the differentiation of the erythropoietin (epo)-producing IW32 erythroleukemia cells was investigated. Podophyllotoxin induced IW32 cells to differentiate, and hemin potentiated the differentiation. Hemin had no effect on cell proliferation whereas podophyllotoxin inhibited cell growth. c-myc mRNA levels decreased biphasically by hemin or podophyllotoxin, while the combined treatment of hemin plus podophyllotoxin did not result in the initial decrease in c-myc mRNA level. Our data suggested that down-regulation of c-myc expression was not a prerequisite of IW32 cell differentiation induced by hemin and podophyllotoxin combined.     

Cloning and expression of the Haemophilus influenzae transferrin receptor genes

The genomic transferrin receptor genes ( tbpA and tbpB  ) from two strains of Haemophilus influenzae type b (Hib) and two strains of non-typable H. influenzae (NTHi) have been cloned and sequenced. The deduced protein sequences of the H. influenzae tbpA genes were 95–100% conserved and those of the tbpB genes were 66–100% conserved. The tbpB gene from one strain of NTHi was found to encode a truncated Tbp2 protein. The tbpB genes from four additional NTHi strains were amplified by the polymerase chain reaction (PCR) utilizing primers derived from the conserved N-terminal sequences of Tbp1 and Tbp2 and were found to encode full-length proteins. Although several bacterial species express transferrin receptors, when the Tbp1 and Tbp2 sequences from different organisms were compared, there was only limited homology. Recombinant Tbp1 and Tbp2 proteins were expressed from Escherichia coli and antisera were raised to the purified proteins. There was significant antigenic conservation of both Tbp1 and Tbp2 amongst H. influenzae strains, as determined by Western blot analysis. In a passive model of bacteraemia, infant rats were protected from challenge with Hib after transfer of anti-rTbp2 antiserum, but not after anti-rTbp1 antiserum.     

Changes in superoxide dismutase, catalase, and the glutathione cycle during induced myeloid differentiation

The human promyelocytic leukemia cell line HL-60 undergoes induced myeloid differentiation, with acquisition of most polymorphonuclear leukocyte (PMN) functions, including generation of toxic oxygen species. We examined the concurrent changes in the cellular detoxifying defenses against superoxide and H2O2: superoxide dismutase, catalase, and the glutathione cycle. During induced differentiation, total superoxide dismutase activity declined to a level slightly more than 2-fold that of PMN, largely due to a decrease in Mn-superoxide dismutase; CuZn-superoxide dismutase showed virtually no change. Catalase activity declined only slightly (but significantly) to a level 1.3 that of PMN. GSH peroxidase activity fell and then rose back to its original level, remaining throughout differentiation more than 10-fold higher than activity in PMN. GSSG reductase activity declined to a level of 73% that of uninduced cells but twice that of PMN. GSH and GSSG contents both decreased, reaching equivalence to those of PMN. Concurrently, the ability of the cells to generate H2O2 increased 11-fold, a change similar to that previously reported for superoxide production. Thus, there is a paradoxical inverse relationship between the development of active oxygen generation and scavenging systems during myeloid differentiation in HL-60 cells.     

Regulated expression of the c-myb and c-myc oncogenes during erythroid differentiation

We have investigated the expression of the genes c-myb, c-myc, and alpha globin in murine erythroid cells at different stages of development, in viral-induced erythroleukemias, as well as in two mouse erythroleukemia cell lines that can be induced to terminally differentiate when exposed to dimethylsulfoxide. We find that there is a reciprocal correlation between the cell's production of messenger RNA for c-myb and globin. c-myc message shows a similar but less dramatic decrease coincident with globin RNA production. Initially with the administration of an inducing agent, dimethylsulfoxide, there is a rapid decrease of myc and myb mRNA, which is followed by signs of differentiation in the induced culture. We conclude that these oncogenes function in early maturational stages of development of these cells. In the erythroleukemic state these genes are down-regulated by forced differentiation and may play a direct role in influencing the state of differentiation of these cells.     

Metabolism of c-myc gene products: c-myc mRNA and protein expression in the cell cycle.

The presence and synthesis of c-myc protein and mRNA in the cell cycle has been studied. We find that c-myc mRNA is present, at equivalent levels, at all times in the cell cycle with the possible exception of mitosis. Furthermore, we demonstrate that this mRNA is transcribed in both G1 and G2 phases. An analysis of the c-myc protein in vivo shows that de novo synthesis occurs in G1 and G2 and the protein turns over with a half-life of approximately 20-30 min in both phases. Furthermore, the level of c-myc protein rapidly increases in cell populations when they re-initiate the cell cycle, thereafter decreasing as the culture reaches quiescence. The results therefore suggest that expression of c-myc can be rapidly modulated and that it is activated during the G0 to G1 transition, but is expressed thereafter in the cell cycle.     

The expression of viral and globin genes during differentiation of the friend cell

A complementary DNA probe has been prepared from the Friend murine erythroleukaemia virus complex (FV) released from Friend cells treated with dimethylsulphoxide (DMSO). The complementary DNA (cDNA) forms a hybrid specifically with the viral RNA genome. The availability of this viral probe together with mouse globin cDNA has made it possible to study the expression of both viral and globin genes in the Friend cell during differentiation using molecular hybridisation techniques. These specific probes have been used in an attempt to determine whether any connection exists between expression of Friend virus sequences and erythroid differentiation as measured by globin gene expression. A titration technique has been used to quantitate the levels of Friend viral- and globin-specific sequences in various Friend cell lines which differ in their ability to release Friend virus in response to DMSO although all produce haemoglobin under the same conditions. The results show: (a) that Friend cell lines unable to release virus nevertheless have a large pool of entire virus specific sequences in the polysomes; (b) an increase in virus release induced with DMSO is normally associated with a modest increase in viral sequence in the polysomes; (c) most cell lines show an early accumulation of viral and a later increase in globin mRNA sequences; (d) in an exceptional virus-negative, BUdR-resistant cell clone (B8/3), the accumulation of globin mRNA takes place very rapidly but there is no concomitant increase in viral RNA during differentiation.     

Relationships between chromosome damage, cell cycle delay and cell killing induced by bleomycin or X-rays

The extent of mitotic delay and chromosome aberration induction by X-rays and bleomycin has been compared in normal human foetal fibroblasts at doses giving approximately equal levels of cell killing, assayed as colony-forming ability. Bleomycin induced much less G2 delay and chromosome damage than X-rays. We conclude that the major mechanism of cell killing by bleomycin does not involve chromosome damage but the cells pass through a number of division cycles before dying and a common DNA lesion is involved in G2 delay and chromosome damage.     

Modulation of the transferrin receptor during DMSO-induced differentiation in HL-60 cells

When HL-60 cells are induced to differentiate by dimethyl sulfoxide along a granulocytic pathway there is a fivefold decrease in the total number of transferrin receptors within 3 days, as compared to untreated cells. This decrease is due primarily to a rapid decline in the synthesis of the receptor rather than an increase in the degradation of the receptor. The decrease in transferrin receptor synthesis is a specific and early event that precedes the cessation of cell proliferation, differentiation, and the decrease in total protein synthesis.     

Chromatin structure of genes during the cell cycle

K562 ceils were separated by centrifugal elutriation into 5 fractions each of which had a unique distribution of ceils at different phases of the cell cycle. A study of DNAseI sensitivity of the e- and -globin genes, which are expressed in these cells, and the light chain immunoglobulin gene, which is not expressed, in nuclei from each of the 5 fractions was undertaken. There was no detectable difference in the arrangement of DNAseI-hypersensitive sites (DHS) in chromatin of each of the three genes in the different fractions.