ALBERT

Description: The colony-stimulating factor-1 (CSF-1) regulates survival, growth, and differentiation of monocytes by binding to a single class of high- affinity receptors. The CSF-1 receptor is identical to the product of the c-fms protooncogene. The present studies monitored the effects of TPA and CSF-1 on c-fms gene expression in human monocytes. The results demonstrate that TPA downmodulates the constitutive expression of c-fms mRNA to low but detectable levels. Treatment of human monocytes with TPA was similarly associated with decreases in levels of the 138- and 125-Kd c-fms-encoded proteins. However, the kinetics of c-fms protein downmodulation indicated independent effects of TPA on c-fms expression at the RNA and protein levels. Furthermore, c-fms protein levels subsequently recovered despite persistently low levels of c-fms mRNA. Although previous studies demonstrated that c-fms protein is down- regulated in the presence of CSF-1, the present results indicate that CSF-1 also downregulates levels of c-fms mRNA. Moreover, the results indicate that CSF-1 increases protein kinase C activity in the membrane fraction. Together, these findings suggest that c-fms gene expression is differentially regulated at both the RNA and protein levels after activation of protein kinase C in human monocytes treated with TPA and CSF-1.

Description: Expression of both the c-fos and c-sis protooncogenes during myeloid differentiation has been detected in cells of the monocytic lineage. Since an increase in c-fos transcripts was not detected during dimethylsulfoxide induced HL-60 granulocytic differentiation, it was suggested that within the myeloid series c-fos gene expression might be lineage specific. In the present study, we have determined whether expression of the c-fos and c-sis genes is indeed specific for the monocytic pathway or rather common to both the granulocyte and monocyte pathways. C-fos and c-sis gene expression was analyzed in freshly isolated human granulocytes and monocytes, in human HL-60 promyelocytic leukemia cells induced to differentiate along the granulocytic or monocytic pathway, in myeloblasts from five patients with the M1 or M2 subtype of acute myeloblastic leukemia (AML) and in blasts from six patients with M4 myelomonocytic leukemia. The level of c-fos mRNA was fifteen times higher in granulocytes as compared with monocytes. An increase in c-fos expression was also found in HL-60 cells differentiated along the granulocytic pathway after exposure to hypoxanthine, hexamethylene bisacetamide, and the combination of retinoic acid and dibutyryl adenosine 3′5′ cyclic monophosphate. Three of 5 M1 and M2 leukemic myeloblast preparations depleted of lymphoid and monocytic cells and all six M4 leukemic cells expressed c-fos transcripts. In contrast, c-sis gene transcripts were detectable in monocytes and during drug induced monocytic differentiation of the HL- 60 cells but not in granulocytes during granulocytic differentiation of the HL-60 cells or in AML samples. Thus, in the myeloid series, c-sis gene expression is lineage specific while expression of the c-fos gene is found in both lineages and may be related to metabolic pathways common to both granulocytes and monocytes.

Description: The murine sarcoma virus 3611 contains the transforming v-raf gene that has partial nucleotide homology with the src family of tyrosine kinase- encoding oncogenes. Although this virus induces fibrosarcomas in mice, a recombinant murine retrovirus carrying both the raf and myc oncogenes induces immunoblastic lymphomas and immortalizes mouse macrophages in vitro. The present study has thus monitored the expression of c-raf in human hematopoietic cells. The results demonstrate the presence of a 3.6-kb c-raf transcript in HL-60 promyelocytic leukemic cells. The induction of HL-60 cell differentiation along the monocytic or granulocytic lineages had no detectable effect on the level of c-raf transcripts. Furthermore, in contrast to c-myc and c-fms expression, inhibition of protein synthesis with cycloheximide had no detectable effect on c-raf expression. Similar levels of c-rafRNA were also found in other human cell lines derived from myeloid, B cell, and T cell tumors, as well as in normal granulocytes, monocytes, and macrophages. These findings suggest that the c-raf protooncogene is widely expressed in multiple hematopoietic lineages.

Description: Phorbol esters induce the human HL-60 promyelocytic cell line to differentiate along a monocytic pathway. This induction of differentiation may involve phorbol ester-induced activation of the phospholipid- and calcium-dependent protein kinase C. Bryostatin 1, a macrocyclic lactone, has been shown to compete with phorbol esters for binding to protein kinase C. We have confirmed that bryostatin 1 translocates activity of protein kinase C from the cytosolic to membrane fractions of HL-60 cells. The present results also demonstrate that bryostatin 1 (10 nmol/L) induces monocytic differentiation of HL- 60 cells as determined by adherence, growth inhibition, appearance of monocyte cell surface antigens, and alpha-naphthyl acetate esterase staining. Furthermore, bryostatin 1 (10 nmol/L) downregulated c-myc expression and induced c-fos, c-fms, and tumor necrosis factor transcripts. These changes in gene expression induced by bryostatin 1 are similar to those associated with phorbol ester-induced monocytic differentiation of HL-60 cells. In contrast, exposure to a higher concentration of bryostatin 1 (100 nmol/L) had less of an effect on growth inhibition of HL-60 cells and changes in gene expression. Moreover, 100 nmol/L bryostatin 1 antagonized the cytostatic effects and adherence induced by phorbol esters. Our results thus suggest that bryostatin 1 activates HL-60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation.

Description: Expression of both the c-fos and c-sis protooncogenes during myeloid differentiation has been detected in cells of the monocytic lineage. Since an increase in c-fos transcripts was not detected during dimethylsulfoxide induced HL-60 granulocytic differentiation, it was suggested that within the myeloid series c-fos gene expression might be lineage specific. In the present study, we have determined whether expression of the c-fos and c-sis genes is indeed specific for the monocytic pathway or rather common to both the granulocyte and monocyte pathways. C-fos and c-sis gene expression was analyzed in freshly isolated human granulocytes and monocytes, in human HL-60 promyelocytic leukemia cells induced to differentiate along the granulocytic or monocytic pathway, in myeloblasts from five patients with the M1 or M2 subtype of acute myeloblastic leukemia (AML) and in blasts from six patients with M4 myelomonocytic leukemia. The level of c-fos mRNA was fifteen times higher in granulocytes as compared with monocytes. An increase in c-fos expression was also found in HL-60 cells differentiated along the granulocytic pathway after exposure to hypoxanthine, hexamethylene bisacetamide, and the combination of retinoic acid and dibutyryl adenosine 3′5′ cyclic monophosphate. Three of 5 M1 and M2 leukemic myeloblast preparations depleted of lymphoid and monocytic cells and all six M4 leukemic cells expressed c-fos transcripts. In contrast, c-sis gene transcripts were detectable in monocytes and during drug induced monocytic differentiation of the HL- 60 cells but not in granulocytes during granulocytic differentiation of the HL-60 cells or in AML samples. Thus, in the myeloid series, c-sis gene expression is lineage specific while expression of the c-fos gene is found in both lineages and may be related to metabolic pathways common to both granulocytes and monocytes.

Description: The colony-stimulating factor-1 (CSF-1) regulates survival, growth, and differentiation of monocytes by binding to a single class of high- affinity receptors. The CSF-1 receptor is identical to the product of the c-fms protooncogene. The present studies monitored the effects of TPA and CSF-1 on c-fms gene expression in human monocytes. The results demonstrate that TPA downmodulates the constitutive expression of c-fms mRNA to low but detectable levels. Treatment of human monocytes with TPA was similarly associated with decreases in levels of the 138- and 125-Kd c-fms-encoded proteins. However, the kinetics of c-fms protein downmodulation indicated independent effects of TPA on c-fms expression at the RNA and protein levels. Furthermore, c-fms protein levels subsequently recovered despite persistently low levels of c-fms mRNA. Although previous studies demonstrated that c-fms protein is down- regulated in the presence of CSF-1, the present results indicate that CSF-1 also downregulates levels of c-fms mRNA. Moreover, the results indicate that CSF-1 increases protein kinase C activity in the membrane fraction. Together, these findings suggest that c-fms gene expression is differentially regulated at both the RNA and protein levels after activation of protein kinase C in human monocytes treated with TPA and CSF-1.

Description: Phorbol esters induce the human HL-60 promyelocytic cell line to differentiate along a monocytic pathway. This induction of differentiation may involve phorbol ester-induced activation of the phospholipid- and calcium-dependent protein kinase C. Bryostatin 1, a macrocyclic lactone, has been shown to compete with phorbol esters for binding to protein kinase C. We have confirmed that bryostatin 1 translocates activity of protein kinase C from the cytosolic to membrane fractions of HL-60 cells. The present results also demonstrate that bryostatin 1 (10 nmol/L) induces monocytic differentiation of HL- 60 cells as determined by adherence, growth inhibition, appearance of monocyte cell surface antigens, and alpha-naphthyl acetate esterase staining. Furthermore, bryostatin 1 (10 nmol/L) downregulated c-myc expression and induced c-fos, c-fms, and tumor necrosis factor transcripts. These changes in gene expression induced by bryostatin 1 are similar to those associated with phorbol ester-induced monocytic differentiation of HL-60 cells. In contrast, exposure to a higher concentration of bryostatin 1 (100 nmol/L) had less of an effect on growth inhibition of HL-60 cells and changes in gene expression. Moreover, 100 nmol/L bryostatin 1 antagonized the cytostatic effects and adherence induced by phorbol esters. Our results thus suggest that bryostatin 1 activates HL-60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation.

Description: The murine sarcoma virus 3611 contains the transforming v-raf gene that has partial nucleotide homology with the src family of tyrosine kinase- encoding oncogenes. Although this virus induces fibrosarcomas in mice, a recombinant murine retrovirus carrying both the raf and myc oncogenes induces immunoblastic lymphomas and immortalizes mouse macrophages in vitro. The present study has thus monitored the expression of c-raf in human hematopoietic cells. The results demonstrate the presence of a 3.6-kb c-raf transcript in HL-60 promyelocytic leukemic cells. The induction of HL-60 cell differentiation along the monocytic or granulocytic lineages had no detectable effect on the level of c-raf transcripts. Furthermore, in contrast to c-myc and c-fms expression, inhibition of protein synthesis with cycloheximide had no detectable effect on c-raf expression. Similar levels of c-rafRNA were also found in other human cell lines derived from myeloid, B cell, and T cell tumors, as well as in normal granulocytes, monocytes, and macrophages. These findings suggest that the c-raf protooncogene is widely expressed in multiple hematopoietic lineages.