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Extensive evidence supports a two-step model for the control of fibroblast growth, which includes first the action of a competence factor (e.g., platelet-derived growth factor) followed by the stimulus of a progression factor (e.g., epidermal growth factor [EGF]). We investigated whether this model may be applied to the euploid EL2 fibroblast line recently isolated from rat embryos (E. Liboi, M. Caruso, and C. Basilico, Mol. Cell. Biol. 4:2925-2928, 1984). Our results clearly show that EGF alone leads EL2 cells to proliferate in serum-free conditions at a rate corresponding to 50 to 60% of that observed in the presence of 10% calf serum. It is of interest that, when resting EL2 cells were exposed to EGF, transcription of both c-myc and c-fos was markedly induced. Altogether, these observations suggest that, in contrast with the model of fibroblast growth mentioned above, EL2 cells require the presence of a single growth factor (EGF) for induction of DNA synthesis, and the expression of myc and fos proto-oncogenes may represent an obligatory step in the pathway of commitment of EL2 cells to proliferation. In addition, we showed that EGF may induce EL2 cells to acquire some properties of transformed cells, such as growth in agar and loss of contact inhibition. This suggests that the particular response to EGF of the EL2 line may be strictly connected with the expression of a transformed phenotype.

We have analyzed the RNA expression of three protein kinase C (PKC) genes (alpha, beta, and gamma) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)+ RNA indicates that expression of PKC alpha and beta genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage, and that the PKC gamma gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)+ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenetic patterns were similar to those observed for human brain. Furthermore, we observed that the expression of PKC gamma is induced in the peri- and postnatal phases. These results suggest that expression of PKC alpha, beta, and gamma genes possibly mediates the development of central neuronal functions, and expression of PKC gamma in particular may be involved in the development of peri- and postnatal functions.

Homeobox (HOX) genes control axial specification during mammalian development and also regulate skin morphogenesis. Although selected HOX genes are variably expressed in leukemias and kidney and colon cancer cell lines, their relationship with the neoplastic phenotype remains unclear. In both normal development and neoplastic transformation, HOX target genes are largely unknown. We investigated the expression and function of HOXB cluster genes in human melanoma. The HOXB7 gene was constitutively expressed in all 25 melanoma cell lines and analyzed under both normal and serum-starved conditions, as well as in in vivo primary and metastatic melanoma cells; conversely, HOXB7 was expressed in proliferating but not quiescent normal melanocytes. Treatment of melanoma cell lines with antisense oligomers targeting HOXB7 mRNA markedly inhibited cell proliferation and specifically abolished expression of basic fibroblast growth factor (bFGF) mRNA. Band shift and cotransfection experiments showed that HOXB7 directly transactivates the hFGF gene through one out of five putative homeodomain binding sites present in its promoter. These novel findings indicate a key role for constitutive HOXB7 expression in melanoma cell proliferation via bFGF. The results also raise the possibility that growth factor genes are critical HOX target genes in other developmental and/or neoplastic cell systems.

Fusion proteins involving the retinoic acid receptor alpha (RAR alpha) and the PML or PLZF nuclear protein are the genetic markers of acute promyelocytic leukemias (APLs). APLs with the PML-RAR alpha or the PLZF-RAR alpha fusion protein are phenotypically indistinguishable except that they differ in their sensitivity to retinoic acid (RA)-induced differentiation: PML-RAR alpha blasts are sensitive to RA and patients enter disease remission after RA treatment, while patients with PLZF-RAR alpha do not. We here report that (i) like PML-RAR alpha expression, PLZF-RAR alpha expression blocks terminal differentiation of hematopoietic precursor cell lines (U937 and HL-60) in response to different stimuli (vitamin D3, transforming growth factor beta1, and dimethyl sulfoxide); (ii) PML-RAR alpha, but not PLZF-RAR alpha, increases RA sensitivity of hematopoietic precursor cells and restores RA sensitivity of RA-resistant hematopoietic cells; (iii) PML-RAR alpha and PLZF-RAR alpha have similar RA binding affinities; and (iv) PML-RAR alpha enhances the RA response of RA target genes (those for RAR beta, RAR gamma, and transglutaminase type II [TGase]) in vivo, while PLZF-RAR alpha expression has either no effect (RAR beta) or an inhibitory activity (RAR gamma and type II TGase). These data demonstrate that PML-RAR alpha and PLZF-RAR alpha have similar (inhibitory) effects on RA-independent differentiation and opposite (stimulatory or inhibitory) effects on RA-dependent differentiation and that they behave in vivo as RA-dependent enhancers or inhibitors of RA-responsive genes, respectively. Their different activities on the RA signalling pathway might underlie the different responses of PML-RAR alpha and PLZF-RAR alpha APLs to RA treatment. The PLZF-RAR alpha fusion protein contains an approximately 120-amino-acid N-terminal motif (called the POZ domain), which is also found in a variety of zinc finger proteins and a group of poxvirus proteins and which mediates protein-protein interactions. Deletion of the PLZF POZ domain partially abrogated the inhibitory effect of PLZF-RAR alpha on RA-induced differentiation and on RA-mediated type II TGase up-regulation, suggesting that POZ-mediated protein interactions might be responsible for the inhibitory transcriptional activities of PLZF-RAR alpha.

The TAL-1gene specifies a basic helix-loop-helix domain (bHLH) transcription factor, which heterodimerizes with E2A gene family proteins. tal-1 protein is abnormally expressed in the majority of T-cell acute lymphoblastic leukemias (T-ALLs). tal-1 is expressed and plays a significant role in normal erythropoietic differentiation and maturation, while its expression in early myeloid differentiation is abruptly shut off at the level of late progenitors/early differentiated precursors (G. L. Condorelli, L. Vitelli, M. Valtieri, I. Marta, E. Montesoro, V. Lulli, R. Baer, and C. Peschle, Blood 86:164–175, 1995). We show that in late myeloid progenitors (the phenotypically normal murine 32D cell line) and early leukemic precursors (the human HL-60 promyelocytic leukemia cell line) ectopic tal-1 expression induces (i) a proliferative effect under suboptimal culture conditions (i.e., low growth factor and serum concentrations respectively), via an antiapoptotic effect in 32D cells or increased DNA synthesis in HL-60 cells, and (ii) a total or marked inhibitory effect on differentiation, respectively, on granulocyte colony-stimulating factor-induced granulopoiesis in 32D cells or retinoic acid- and vitamin D3-induced granulo- and monocytopoiesis in HL-60 cells. Furthermore, experiments with 32D temperature-sensitive p53 cells indicate that aberrant tal-1 expression at the permissive temperature does not exert a proliferative effect but causes p53-mediated apoptosis, i.e., the tal-1 proliferative effect depends on the integrity of the cell cycle checkpoints of the host cell, as observed for c-mycand other oncogenes. tal-1 mutant experiments indicate that ectopic tal-1 effects are mediated by both the DNA-binding and the heterodimerization domains, while the N-terminally truncated tal-1 variant (M3) expressed in T-ALL malignant cells mimics the effects of the wild-type protein. Altogether, our results (i) indicate proliferative and antidifferentiative effects of ectopic tal-1 expression, (ii) shed light on the underlying mechanisms (i.e., requirement for the integrity of the tal-1 bHLH domain and cell cycle checkpoints in the host cell, particularly p53), and (iii) provide new experimental models to further investigate these mechanisms.

Zinc finger genes encode proteins that act as transcription factors. The myeloid zinc finger 1 (MZF1) gene encodes a zinc finger protein with two DNA-binding domains that recognize two distinct consensus sequences, is preferentially expressed in hematopoietic cells, and may be involved in the transcriptional regulation of hematopoiesis-specific genes. Reverse transcription-PCR analysis of human peripheral blood CD34+ cells cultured under lineage-restricted conditions demonstrated MZF1 expression during both myeloid and erythroid differentiation. Sequence analysis of the 5'-flanking region of the CD34 and c-myb genes, which are a marker of and a transcriptional factor required for hematopoietic proliferation and differentiation, respectively, revealed closely spaced MZF1 consensus binding sites found by electrophoretic mobility shift assays to interact with recombinant MZF1 protein. Transient or constitutive MZF1 expression in different cell types resulted in specific inhibition of chloramphenicol acetyltransferase activity driven by the CD34 or c-myb 5'-flanking region. To determine whether transcriptional modulation by MZF1 activity plays a role in hematopoietic differentiation, constructs containing the MZF1 cDNA under the control of different promoters were transfected into murine embryonic stem cells which, under defined in vitro culture conditions, generate colonies of multiple hematopoietic lineages. Constitutive MZF1 expression interfered with the ability of embryonic stem cells to undergo hematopoietic commitment and erythromyeloid colony formation and prevented the induced expression of CD34 and c-myb mRNAs during differentiation of these cells. These data indicate that MZF1 plays a critical role in hematopoiesis by modulating the expression of genes involved in this process.