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BMI1 is a key component of the PRC1 (polycomb repressive complex-1) complex required for maintenance of normal and cancer stem cells. Its aberrant expression is detected in chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia (ALL), but no data exist on BMI1 requirement in ALL cells. We show here that BMI1 expression is important for proliferation and survival of Ph+ ALL cells and for leukemogenesis of Ph+ cells in vivo. Levels of BIM, interferon-a (IFNa)-regulated genes and E2F7 were upregulated in BMI1-silenced cells, suggesting that repressing their expression is important for BMI1 biological effects. Consistent with this hypothesis, we found that: (i) downregulation of BIM or E2F7 abrogated apoptosis or rescued, in part, the reduced proliferation and colony formation of BMI1 silenced BV173 cells; (ii) BIM/E2F7 double silencing further enhanced colony formation and in vivoleukemogenesis of BMI1-silenced cells; (iii) overexpression of BIM and E2F7 mimicked the effect of BMI1 silencing in BV173 and SUP-B15 cells; and (iv) treatment with IFNa suppressed proliferation and colony formation of Ph+ ALL cells. These studies indicate that the growth-promoting effects of BMI1 in Ph+ ALL cells depend on suppression of multiple pathways and support the use of IFNa in the therapy of Ph+ ALL.

Using the representation difference analysis technique, we have identified a novel gene, Ian4, which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the Bcr/Abl oncogene. Ian4 expression was undetectable in 32D cells transfected with v-src, oncogenic Ha-ras or v-Abl. Murine Ian4 maps to chromosome 6, 25 cM from the centromere. The Ian4 mRNA contains two open reading frames (ORFs) separated by 5 nt. The first ORF has the potential to encode for a polypeptide of 67 amino acids without apparent homology to known proteins. The second ORF encodes a protein of 301 amino acids with a GTP/ATP-binding site in the N-terminus and a hydrophobic domain in the extreme C-terminus. The IAN-4 protein resides in the mitochondrial outer membrane and the last 20 amino acids are necessary for this localization. The IAN-4 protein has GTP-binding activity and shares sequence homology with a novel family of putative GTP-binding proteins: the immuno-associated nucleotide (IAN) family.

Nm23 gene family has been associated with metastasis suppression and differentiation. We studied DR-nm23 during neuroblastoma cells differentiation. DR-nm23 expression increased after retinoic acid induction of differentiation in human cell lines SK-N-SH and LAN-5. In several cell lines, overexpression of DR-nm23 was associated with more differentiated phenotypes. SK-N-SH cells increased vimentin expression, increased deposition of collagen type IV, modulated integrin expression, and underwent growth arrest; the murine neuroblastoma cell line N1E-115 showed neurite outgrowth and a striking enhancement of β1 integrin expression. Up-regulation of β1 integrin was specifically responsible for the increase in the adhesion to collagen type I-coated plates. Finally, cells overexpressing DR-nm23 were unable to growth in soft agar. In conclusion, DR-nm23 expression is directly involved in differentiation of neuroblastoma cells, and its ability to affects the adhesion to extracellular substrates and to inhibit growth in soft agar suggests an involvement in the metastatic potential of neuroblastoma. Med. Pediatr. Oncol. 36:93–96, 2001. © 2001 Wiley-Liss, Inc.

These studies aimed to determine the expression and functional role of c-mybin erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic-fetal liver (FL) were treated with either c-mybantisense oligomers or 3H-thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myboligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-mybon the number of BFU-E colonies was 28.3% ± 15.8% in PB, 53.4% ± 9.3% in BM, and 68.2% ± 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited (73.2% ± 10.4% and 74.2% ± 12.7%). Using highly purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% ± 4.2% in PB, 29.4% ± 6.5% in BM, and 40.1% ± 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% ± 7.9% and 60.98% ± 6.6%, respectively. We then investigated the kinetics of c-mybmRNA level during the erythroid differentiation of highly purified adult PB and FL BFU-E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-mybmRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-mybmRNA and protein in differentiating embryonic precursors peaked 2 days earlier. In both cases, c-mybmRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-mybantisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase. Under the same experimental conditions a progressive increase of the mRNA level of DNA polymerase α was observed. These observations suggest that in early erythroid differentiation c-mybactivation is associated with the progression of progenitors into the S phase of the cell cycle, as well as to the synthesis of DNA polymerase α

These studies aimed to determine the expression and functional role of c-myb in erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU- E) and colony-forming unit (CFU-E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic-fetal liver (FL) were treated with either c-myb antisense oligomers or 3H-thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myb oligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-myb on the number of BFU-E colonies was 28.3% +/- 15.8% in PB, 53.4% +/- 9.3% in BM, and 68.2% +/- 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited (73.2% +/- 10.4% and 74.2% +/- 12.7%). Using highly purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% +/- 4.2% in PB, 29.4% +/- 6.5% in BM, and 40.1% +/- 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% +/- 7.9% and 60.98% +/- 6.6%, respectively. We then investigated the kinetics of c-myb mRNA level during the erythroid differentiation of highly purified adult PB and FL BFU-E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-myb mRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-myb mRNA and protein in differentiation embryonic precursors peaked 2 days earlier. In both cases, c-myb mRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-myb antisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase. Under the same experimental conditions a progressive increase of the mRNA level of DNA polymerase alpha was observed. These observations suggest that in early erythroid differentiation c-myb activation is associated with the progression of progenitors into the S phase of the cell cycle, as well as to the synthesis of DNA polymerase alpha.

The CD34 cell surface antigen is a glycoprotein expressed by hematopoietic stem and progenitor cells and also by certain nonhematopoietic cell-types. Because CD34 expression is regulated both at the transcriptional and the posttranscriptional level, we attempted to identify factors that, by interacting with the 5′ flanking region of the human CD34 gene, may regulate its promoter activity in proliferating hematopoietic cells. By electrophoretic mobility shift assay, UV cross-linking and DNase I footprinting analyses, we identified a multiprotein complex, designated NC-3A, that specifically interacts with the CD34 promoter region from nucleotides -375 to -351. Sequence analysis of this region revealed the presence of a distinct motif, TCATTT. Chloramphenicol acetyl-transferase assays used to assess promoter activity in transiently transfected cells showed that this TCATTT-containing element, which is conserved in both the human and the murine CD34 genes, mediates positive regulatory activity in hematopoietic and nonhematopoietic cells, and acts as an enhancer when placed upstream of a heterologous promoter. Moreover, loss of CD34 promoter activity was caused by mutation of the TCATTT motif. In addition, the interaction of the nuclear multiprotein complex NC-3A with this enhancer element is proliferation-dependent. These data indicate that, although not cell-type specific, the formation of a multiprotein complex NC-3A interacting with the region from nucleotides -375 to 351 plays an important role in controlling CD34 promoter activity in proliferating hematopoietic cells.

The proliferation of chronic myelogenous leukemia (CML) cells and the transformation of normal hematopoietic cells by BCR-ABL appear to require the expression of a functional MYC protein, suggesting an approach to treatment of Philadelphia leukemias based on simultaneous targeting of BCR-ABL and c-MYC. To test this hypothesis, CML-blast crisis (CML-BC) primary cells were treated in vitro with bcr-abl and c- myc antisense phosphorothioate oligodeoxynucleotides ([S]ODNs), individually or in combination. Compared with antisense ODNs targeting of individual oncogenes, downregulation of both BCR-ABL and c-MYC by specific antisense [S]ODNs resulted in a synergistic antiproliferative effect. Colony formation of normal bone marrow cells was not affected by either treatment. To assess the therapeutic potential of multiple oncogene downregulation, SCID mice injected with CML-BC primary cells were treated systematically with equal doses of bcr-abl or c-myc antisense [S]ODNs or with a combination of both antisense [S]ODNs. Compared with mice treated with individual compounds, the disease process was significantly retarded in the group treated with both [S]ODNs as revealed by flow cytometry, clonogenic assay, and RT-PCR analysis to detect leukemic cells in mouse tissue cell suspensions. These effects correlated with a markedly increased survival of leukemic mice treated with both antisense [S]ODNs. Leukemic cells harvested from antisense [S]ODN-treated mice were sensitive to the effects of antisense [S]ODNs in vitro, suggesting that the treatment can be successfully repeated. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.

Overexpression of wild-type p53 gene in malignant cell lines has been shown to inhibit cell proliferation in a number of cases. However, endogenous p53 protein seems to play little role in normal cell-cycle control as suggested by the normal development of p53 null mice, and by the low p53 protein levels expressed in most cell types. Recently, increased expression of endogenous p53 protein has been observed during the cellular response to DNA damage, as well as during differentiation of human hematopoietic cells. To study the role of the p53 gene in hematopoietic differentiation, we introduced the wild-type p53 gene or the temperature-sensitive p53(Val135) mutant into p53-deficient HL-60 promyelocytic leukemia cells. Morphological analysis, flow-cytometric determination of granulocytic or monocytic surface markers, and ability to reduce nitroblue tetrazolium (NBT) demonstrated that expression of exogenous wild-type p53 gene in HL-60 cells induces differentiation through the granulocytic pathway. Proliferation and cell-cycle analysis performed early after expression of wild-type p53 showed that induction of differentiation is not coupled with growth arrest, which suggests that p53 is involved in differentiation independently of its activity on the cell cycle.

We have found that administration of chemotherapy alters expression of growth-regulated genes in leukemia blast cells. To determine if such changes might be correlated with therapeutic outcome, we studied steady-state mRNA levels of MYC and histone H3 in the leukemic blasts of patients just prior to and 24 hr after the administration of the first doses of antileukemic drug therapy. Among nine patients with acute myelogenous leukemia, mRNA levels of MYC and histone H3 were reduced in five patients, and hematologic remission was achieved in three of these individuals. No remission was obtained in the four patients without reduction in MYC and histone H3 mRNA. Among acute lymphocytic leukemia patients, the mRNA levels of MYC and/or histone H3 were reduced by the therapy in seven of nine patients. A complete hematologic remission was obtained in five of them, and a partial remission was obtained in the other two. No remission was obtained in the patients in which MYC and H3 mRNA levels were unaffected by the therapy. These studies are of interest because they suggest that a decrease in the mRNA levels of MYC and histone H3 24 hr after a single dose of antineoplastic drugs may predict which patients will achieve complete remission; lack of reduction in these mRNAs correlates with failure to achieve remission. In addition, these studies also provide further proof of the heterogeneity of altered growth regulation among human leukemias.

We have established the complete coding sequence of the human vimentin gene. It had 91% homology to the coding sequence of the Syrian hamster vimentin gene (Quax et al., Cell 35:215-223, 1983) and partial homology to several other sequences coding for intermediate filament proteins. The most striking difference between the Syrian hamster and human vimentin genes was in the 3' untranslated region, which was considerably longer in the Syrian hamster. Using RNA blots and a human vimentin cDNA clone from an Okayama-Berg library, we have established that expression of the vimentin gene was growth regulated. The steady-state levels of cytoplasmic vimentin mRNA in 3T3 cells were increased by serum and platelet-derived growth factor, but not by epidermal growth factor, insulin, or platelet-poor plasma. The increase in expression of the vimentin gene that occurred when G0-phase cells were stimulated to proliferate was detected in six different cell types from four different species. The expression of the vimentin gene was also increased when HL60 cells were induced to differentiate by phorbol esters; it decreased when differentiation was induced by retinoic acid.

Total RNA extracted from peripheral blood lymphocytes of a patient with B-cell chronic lymphocytic leukemia (CLL) and the poly (A+) RNA was purified. A cDNA library was constructed and approximately 4,000 clones were screened in order to identify genes preferentially expressed in CLL. A relatively low repetition frequency characterizes the majority of the abundant mRNA species present in CLL lymphocytes. One clone, corresponding to the mRNA encoding the HLA-DR-associated invariant chain, was selected and its expression was examined in different leukemic cell populations and in normal tissues. DNA-RNA hybridization studies showed that the invariant chain mRNA (In-mRNA) is detectable in RNA preparations from human blood cells and their precursors, whereas no In-mRNA is found in several other tissues examined. Among various normal and leukemic leukocyte populations, the highest levels of In- mRNA are found in CLL. Therefore, a role of In-chain mRNA as a marker of CLL is proposed. Our data support a relationship between high levels of invariant chain mRNA and the out of cycle condition of CLL peripheral blood lymphocytes.

Chronic myelogenous leukemia evolves in two clinically distinct stages: a chronic and a blast crisis phase. The molecular changes associated with chronic phase to blast crisis transition are largely unknown. We have identified a cDNA clone, DR-nm23, differentially expressed in a blast-crisis cDNA library, which has approximately 70% sequence similarity to the putative metastatic suppressor genes, nm23-H1 and nm23-H2. The deduced amino acid sequence similarity to the proteins encoded by these two latter genes is approximately 65% and includes domains and amino acid residues (the leucine zipper-like and the RGD domain, a serine and a histidine residue in the NH2- and in the COOH-terminal portion of the protein, respectively) postulated to be important for nm23 function. DR-nm23 mRNA is preferentially expressed at early stages of myeloid differentiation of highly purified CD34+ cells. Its constitutive expression in the myeloid precursor 32Dc13 cell line, which is growth-factor dependent for both proliferation and differentiation, results in inhibition of granulocytic differentiation induced by granulocyte colony-stimulating factor and causes apoptotic cell death. These results are consistent with a role for DR-nm23 in normal hematopoiesis and raise the possibility that its overexpression contributes to differentiation arrest, a feature of blastic transformation in chronic myelogenous leukemia.

A genomic DNA library consisting of human DNA fragments about 18 kilobases long cloned in a bacteriophage lambda vector was found to contain a specific repeated DNA segment. The repeated sequence is present in greater than 95% of the genomic library, and selected clones contain at least two copies of the sequence. Our experiments indicate that this highly repetitive sequence (approximately 400,000 copies per haploid genome) is widely distributed in the human genome and is represented in the cytoplasmic polysomal mRNA. This sequence is homologous to the 300-base-pair Alu repeat family, the predominant repeat sequence in man.

Transformation of hematopoietic cells by the p210bcr/abl tyrosine kinase appears to require the expression of a functional MYC protein, suggesting that simultaneous targeting of BCR-ABL and c-myc might be a rational strategy for attempting treatment of Phil-adelphia leukemia. To test this hypothesis, severe combined immunodeficiency mice injected with Philadelphia leukemic cells were treated systemically with equal doses of bcr-abl or c-myc antisense oligodeoxynucleotides (ODNs) or with both ODNs in combination. Compared with the mice treated with individual agents, the disease process was much slower in the group treated with both ODNs, as revealed by flow cytometry, clonogenic assay, and reverse transcriptase-polymerase chain reaction analysis to detect leukemic cells in mouse tissue cell suspensions, and by enumeration of liver metastases. The retardation of the disease process was positively correlated with a markedly increased survival of leukemic mice treated with both ODNs. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.

Heterogeneous nuclear RNA was extracted from normal PHA-stimulated human lymphocytes and acute myeloid leukemia blast cells. Experiments were performed to determine the hybridization kinetics of these RNAs to human DNA. The best least squares solutions indicate in the hybridization reaction of both normal and leukemic RNA two main components. For leukemic cell RNA the rate constants of both components were significantly different from that of normal cell RNA. In particular, the difference between the rate constants of the second slower component suggests that the slowly hybridizing sequences in leukemic cell RNA have a degree of repetition higher than that of the corresponding sequences of normal cell RNA.

The retinoblastoma protein family has been implicated in growth control and modulation of the activity of genes involved in cell proliferation, such as B-myb. Recent evidence indicates that the product of the B-myb gene is necessary for the growth and survival of several human and murine cell lines. Upon overexpression, B-myb induces deregulated cell growth of certain cell lines. Here we show that B-myb overexpression is able to induce DNA synthesis in p107 growth-arrested human osteosarcoma cells (SAOS2). p107 might exert its growth-suppressive activity by regulating B-myb gene transcription. Indeed, p107 down-modulated B-myb promoter activity and drastically decreased E2F-mediated transactivation. Finally, B-myb was able to stimulate DNA synthesis of both stably and transiently transfected human glioblastoma cells (T98G). Altogether, these data provide definitive evidence that the human B-myb protein is involved in growth control of human cells, and that p107 has a significant role in regulating B-myb gene activity.

The enzymes of the DNA synthesizing machinery constitute a group of gene products that are generally expressed co-ordinately at the G1/S boundary of the cell cycle. We have investigated how growth factors regulate the steady-state mRNA levels of two of these genes, the PCNA (proliferating cell nuclear antigen)/cyclin and the thymidine kinase genes. To detect the PCNA/cyclin mRNA, we isolated a cDNA clone from a human library. Two different cell lines were used for these studies: BALB/c3T3 cells, which are exquisitely sensitive to growth factors, and ts13 cells, a temperature-sensitive (ts) mutant of the cell cycle, which arrests in G1 at the restrictive temperature. The steady-state levels of the RNAs for these two genes under different growth conditions were also compared with the levels of histone H3 RNA which are good indicators of the fraction of cells in S phase. Both PCNA/cyclin and thymidine kinase genes share two fundamental characteristics, i.e. they are not inducible in a G1-specific ts mutant of the cell cycle at the restrictive temperature and their expression is inhibited by cycloheximide, indicating that unlike early growth-regulated genes, they require the previous expression of other growth-regulated genes. However, the two genes also show differences, the most notable being that PCNA/cyclin is inducible by epidermal growth factor alone, while thymidine kinase is not.

Exposure of human leukemia HL-60 cells to an oligodeoxynucleotide complementary to an 18-base sequence (codons 2-7) of c-myb-encoded mRNA has previously been shown to result in inhibition of cell proliferation. Because HL-60 cells express high levels of transferrin receptor we adapted a DNA delivery system based on receptor-mediated endocytosis to introduce myb oligomers complexed with a transferrin-polylysine conjugate into those cells. A DNA.RNA duplex resistant to S1 nuclease digestion was detected as early as 12 hr after culture of HL-60 cells in the presence of the myb antisense/transferrin-polylysine complex. Exposure of HL-60 cells to the myb antisense/transferrin-polylysine complex resulted in rapid and profound inhibition of proliferation and loss of cell viability much more pronounced than that occurring in cells exposed to free myb antisense oligodeoxynucleotides. The transferrin-polylysine/myb sense complex or the transferrin-polylysine conjugate alone had no effect on HL-60 cell proliferation and viability. These findings indicate that myb synthetic oligodeoxynucleotides enter efficiently into HL-60 by transferrin receptor-mediated endocytosis and exert a profound biological effect. Such a delivery system could exploit other ligand-receptor interactions for the selective delivery of oncogene-targeted antisense oligodeoxynucleotides.

A number of genes and cDNA sequences (including at least four oncogenes) are known to be expressed in a cell cycle-dependent manner, i.e. the levels of specific mRNAs vary with the phases of the cell cycle. In order to explore the significance of some of these sequences in the mitogenic response, we have investigated the expression of 8 cell cycle-dependent sequences (plus two control sequences, not expressed in a cell cycle-dependent manner) under a variety of conditions. These conditions included cells of different types, from different species, stimulated to proliferate by different mitogens. The genes (or sequences) studied included five cDNA clones whose sequences are preferentially expressed in early G1, i.e. two cDNA clones inducible by platelet-derived growth factor (JE-3 and KC-1), and three cDNA clones inducible by serum (2A9, 2F1, 4F1); and three oncogenes (c-myc, c-rasHa and p53) whose expression is known to be cell cycle-dependent. All of the tested genes, except 2A9, c-rasHa and the control genes, are expressed in a cell cycle-dependent manner in human peripheral blood mononuclear cells stimulated by phytohemagglutinin and in serum-stimulated mouse and Syrian hamster fibroblasts. The inducibility of these genes by different mitogens in cells of different types and from different species strongly suggests that these genes play a role in cell cycle progression. This conclusion is further supported by the known structural and functional similarities between cell-cycle dependent genes, oncogenes and genes coding for cell-cycle related molecules.

We have identified a cDNA whose sequence is preferentially expressed when quiescent fibroblasts are stimulated to proliferate. The steady-state levels of the mRNA corresponding to this clone, called 2A9, are increased by serum, platelet-derived growth factor, and epidermal growth factor, but not by insulin or platelet-poor plasma. mRNA levels of 2A9 are also increased in human acute myeloid leukemia. The 2A9 cDNA has been molecularly cloned from an Okayama-Berg library, and its complete nucleotide sequence has been determined. It has an open reading frame of 270 nucleotides, which has a 55% homology with the coding sequence of the beta-subunit of the S-100 protein, a calcium-binding protein that belongs (like calmodulin and the vitamin D-dependent intestinal calcium-binding protein) to the family of calcium-modulated proteins and is found in abundance in several human tumors, including melanoma. The S-100 protein and the deduced aminoacid sequence of 2A9 are also partially homologous to the small subunit of a protein complex that serves as a cellular substrate to tyrosine kinase. The partial homology of 2A9 (whose RNA is inducible by growth factors and is overexpressed in human acute myeloid leukemias) to the S-100 protein, other calcium-modulated proteins, and the subunit of a substrate for tyrosine kinase, is particularly interesting in view of the role attributed to calcium and tyrosine kinases in the regulation of cell proliferation.

The rapid progress in the cloning of proteoglycan genes has enabled investigators to examine in depth the functional roles these polyhedric molecules play in the control of cell proliferation. Decorin, a leucine-rich proteoglycan expressed by most connective tissues, is a prototype molecule that regulates cellular growth via two mechanisms: modulation of growth factor activity and matrix assembly. We now provide direct evidence that human colon cancer cells stably transfected with decorin cDNA exhibit a marked suppression of the transformed phenotype: the cells have a reduced growth rate in vitro, form small colonies in soft agar, and do not generate tumors in scid/scid mice. Several independent clones are arrested in the G1 phase of the cell cycle, and their growth suppression can be restored by treatment with decorin antisense oligodeoxynucleotides. These effects are independent of growth factors and are not due to either clonal selection or integration site of the decorin gene. These findings correlate well with the observation that decorin gene expression is markedly up-regulated during quiescence. Decorin thus appears to be one component of a negative loop that controls cell growth.

The inhibitory effect of c-myb antisense oligodeoxynucleotides (ODNs) conjugated to folic acid (FA) on HL-60 cell proliferation was examined. Folic acid was covalently linked to a polylysine chain and purified by gel chromatography. Sterile FA-polylysine was complexed with c-myb sense and antisense. Exposure of HL-60 cells to the FA-polylysine-c-myb antisense ODN complex resulted in a down-regulation of c-myb expression and a greater inhibition of proliferation than that obtained using free ODNs. Moreover, FA-polylysine conjugate alone or complexed to c-myb sense ODN was not toxic to cells. The antigenic properties and uptake of the vitamin were not affected by the polylysine chain. These data suggest that this strategy is potentially useful for the selective delivery of anti-oncogene-targeted ODNs into cancer cells.

The expression of a hsp70 gene in human cells has previously been shown to be related to the growth state of the cells. As an alternative to in vitro synchronization procedures, we have measured steady-state levels of the RNA for a heat-shock protein 70 (hsp70) in human peripheral blood mononuclear cells (PBMC) that are naturally quiescent in a G0 state. The probe used recognized, on RNA blots, one single band. The levels of this hsp70 RNA are elevated in circulating PBMC and decrease when the cells are incubated with serum, or phytohemagglutinin, or simply when they are incubated in culture medium. The levels of hsp70 RNA decrease within 30 min after in vitro culture, and are accompanied by an increase in the levels of c-fos RNA. These findings, together with other recent reports in the literature, suggest a possible role of the hsp70 proteins in the regulation of cell growth.

We have investigated the expression of certain cell-cycle-dependent genes in human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin (PHA). The genes studied had been previously identified as cell-cycle dependent in other cell types from different species and were induced by different mitogens. One of these genes (2F1) and the gene for the interleukin 2 receptor were induced by PHA even in cultures partially depleted of accessory cells where the lymphocytes grew in size but failed to enter S phase. The other genes (c-myc, 4F1, JE-3, and KC-1) were induced only in complete cultures of PBMC stimulated by PHA. These results confirm the dissociation between growth in size and cell DNA replication that can occur during cell-cycle progression. Moreover, the time course of appearance of detectable levels of RNA for these genes suggests that they may be used as markers of cell-cycle progression in the transition of lymphocytes from G0 to S phase.

The proliferating cell nuclear antigen (PCNA, cyclin) was originally defined as a nuclear protein whose appearance correlated with the proliferative state of the cell. It is now known to be a co-factor of DNA polymerase δ and to be necessary for DNA synthesis and cell cycle progression. cDNA clones of human PCNA have been isolated and, using one of these cDNA, we have now obtained from a λ phage library a clone containing the entire human PCNA gene and flanking sequences. The human PCNA gene is a unique copy gene and has 6 exons. It spans, from the cap site to the poly(A) signal 4961 base pairs. We have identified, in the 5′-flanking sequence, a region with promoter activity, a well as other structural elements common to other promoters. An interesting feature of the PCNA gene is the presence of extensive sequence similarities among introns and between introns and exons.

CD34 is currently the only well defined human hematopoietic stem cell marker and is expressed on 1-4& of normal bone marrow cells. Putative binding sites for Ets proteins, a family of transcription factors involved in the regulation of cell differentiation and proliferation in many cell systems, are present in the 5'-flanking region of the CD34 gene. Some of these sites are in close proximity to binding sequences of the encoded product of the proto-oncogene c-myb, which regulates CD34 expression by interacting with the Myb binding sites. Here we demonstrate that Ets-2 (i) transactivates the CD34 promoter in rodent fibroblasts upon interaction with Ets binding sites and (ii) induces expression of CD34 mRNA and protein in the CD34- human glioblastoma T98G cells. Ets-2 and c-Myb transactivate the CD34 promoter independently because specific transactivation is abrogated by site-specific mutations of the binding sites or by competition with oligomers that include wild type but not mutated Myb or Ets binding sites. Ets-2 and c-Myb appear to have addictive effects on transactivation of the CD34 promoter and on induction of CD34 mRNA. Instead, CD34 surface protein levels might be induced synergistically, raising the possibility of a posttranslational mechanism of CD34 expression in cells constitutively expressing c-Myb and Ets-2.

The B-myb gene is expressed in many cell types at the G1/S transition of the cell cycle. Inhibition of B-myb expression in BALB/c 3T3 fibroblasts by introduction of a B-myb antisense construct greatly diminished cell proliferation, whereas constitutive expression of a human B-myb cDNA in these cells reduced their growth factor requirements and induced a transformed phenotype. Constitutive expression of B-myb cDNA was accompanied by activation of cyclin D1 and cdc2 expression but not of cyclin A and cyclin B. Transfection of BALB/B-myb cells (a cell line expressing high levels of exogenous human B-myb) with a cyclin D1 antisense construct drastically reduced cloning efficiency of these cells. These results suggest that the B-myb-encoded product regulates fibroblast proliferation by activating cdc2 and cyclin D1 gene expression and that abnormal expression of cyclin D1 might be a step in the process of transformation.

The c-myb protooncogene is preferentially expressed in hematopoietic cells, and its encoded protein, Myb, is required for hematopoietic cell proliferation. To analyze the relative Myb dependence of normal and leukemic human hematopoietic progenitor cells, normal bone marrow cells, several types of leukemic blast cells, and 1:1 mixtures of normal and leukemic cells were cultured in the presence of c-myb sense or antisense oligodeoxynucleotides; cell viability and cloning efficiency were then assessed. c-myb sense oligomers had negligible effects on normal and leukemic cells. In contrast, c-myb antisense oligomers strongly inhibited or completely abolished clonogenic growth of a T-cell leukemia line, 78% (18 of 23) of primary acute myelogenous leukemia cases examined, and 4 of 5 primary chronic myelogenous leukemia (CML) cases in blast crisis. In three of the latter patients, polymerase chain reaction analysis of a 1:1 mixture of c-myb antisense-treated normal and CML cells revealed a complete absence of bcr-abl expression, suggesting that the CML clonogenic units had been completely eliminated from the cultures. At antisense doses that inhibited leukemic cell growth, normal hematopoietic progenitor cells survived. Thus, normal and leukemic hematopoietic cells show differential sensitivity to the toxic effects of c-myb antisense DNA. Perturbation of c-myb function with antisense oligodeoxynucleotides might eventually form the basis for a molecular approach to leukemia therapy, perhaps most immediately as ex vivo bone marrow purging agents.

We recently found that inhibition of MYB protein synthesis in human peripheral blood mononuclear cells (PBMC) exposed to human c-myb (designated MYB) antisense oligodeoxynucleotides prevents entry into S phase and cell proliferation. To determine the mechanism(s) by which down-regulation of human c-myb protein (MYB) synthesis interferes with DNA synthesis, we analyzed mRNA levels of DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), transcripts of two genes required for DNA synthesis, in normal and leukemic T lymphocytes exposed to MYB antisense oligodeoxynucleotides. Expression of DNA polymerase alpha was inhibited both in normal T lymphocytes progressing from G0 to S phase and in exponentially growing CCRF-CEM leukemic cells, whereas expression of PCNA was inhibited only in mitogen-stimulated PBMC and remained essentially unaffected in the leukemia T-cell line. The functional link between expression of MYB and DNA polymerase alpha mRNAs was further demonstrated by analyzing DNA polymerase alpha mRNA levels in a temperature-sensitive (ts) fibroblast cell line (TK-ts13; TK is thymidine kinase) constitutively expressing human MYB mRNA driven by the simian virus 40 (SV40) promoter. In the MYB-expressing TK-ts13 cells, DNA polymerase alpha mRNA levels were unaffected following shift to the nonpermissive temperature of 39.6 degrees C, whereas in the parental line, DNA polymerase alpha mRNA levels were readily down-regulated. These findings indicate that the expression of MYB is related to that of DNA polymerase alpha in cells expressing MYB at high levels and suggest that there is a functional link between c-myb and DNA polymerase alpha mRNA expression during cell cycle progression of normal T lymphocytes.

Approximately one-half of the polysomal poly(A)+RNA from CCRF-CEM human lymphoblastoid cells associates at low R0t (10 M.sec) [where R0 is the initial concentration of RNA (M) and t is time (sec)] to form branched complexes detectable by electron microscopy. The complexes typically involve 2-16 molecules associated over double-stranded regions 120 +/- 30 base pairs long. Formation of such complexes suggests that poly(A)+RNA contains repeated-sequence elements that are highly represented in the mRNA population. Hybridization of polysomal poly(A)+RNA with a recombinant human DNA plasmid, p lambda H15C, which is shown to contain at least three regions complementary to two different members of the Alu family of DNA repeat sequences, showed a total of five regions where R loops are formed. The hybridized regions comprising these groups are 260 +/- 180, 240 +/- 170, 150 +/- 70, 180 +/- 60, and 180 +/- 80 base pairs long. The relative frequencies of R loops formed at these different sites indicate that sequences in this recombinant DNA are represented in the mRNA population at different frequencies. The hybridizing sequence of the RNA molecules is located near one terminus in 13% of the R loops and internally in 53% of the R loops. Surprisingly, 35% of the R loops apparently involve RNA molecules hybridized over their entire length of only 200 +/- 110 base pairs.

Treatment of five human myeloid leukemic cell lines (KG1, ML3, HL-60, U-937, and HEL) with TPA was followed by macrophage differentiation and was accompanied by an early and transient increase in the mRNA level of c-fos proto-oncogene. The induction of c-fos was also observed in human cell lines K562 and K-Gla that did not respond to TPA with terminal macrophage differentiation. The treatment of HL-60 and U-937 cell lines with 1-oleoyl-2-acetylglycerol, a synthetic analog of diacylglycerol that, like TPA, stimulates protein kinase C activity, was followed by early and transient induction of c-fos mRNA in the absence of terminal macrophage differentiation. Finally, treatment of HL-60 with TPA in the presence of retinal, an inhibitor of protein kinase C, drastically reduced the induction of c-fos mRNA but had no effect on the terminal macrophage differentiation that is induced in this cell line by TPA. These results indicate that the induction of c-fos and terminal macrophage differentiation in response to TPA treatment can be dissociated in the in vitro models provided by human myeloid leukemic cell lines. Moreover, these findings suggest that the induction of c-fos is not only insufficient but may also be unnecessary for the differentiation along the monocyte-macrophage pathway.

There is evidence to suggest that the p120 GAP (GAP), originally described as an inhibitor of p21ras, may also serve as a downstream effector of ras-regulated signal transduction. To determine whether GAP expression is required for the growth of human normal and leukemic hematopoietic cells, we used GAP antisense oligodeoxynucleotides to inhibit it and analyzed the effects of this inhibition on the colony-forming ability of nonadherent, T lymphocyte-depleted mononuclear cells and of highly purified progenitors (CD34+ MNC) obtained from the bone marrow and peripheral blood of healthy volunteers or chronic myeloid leukemia (CML, bcr-abl-positive) patients. The acute myelogenous leukemia cell line MO7, the Philadelphia BV173 cell line, and the acute promyelocytic leukemia NB4 and HL-60 cell lines were similarly examined. GAP antisense treatment inhibited colony formation from normal myelo-, erythro-, and megakaryopoietic progenitor cells as well as from CML progenitor cells. Proliferation of MO7 (growth factor-dependent) and BV173 (bcr-abl-dependent) cells, but not that of NB4 and HL-60 (growth factor-independent) cells, was also inhibited, even though a specific downregulation of GAP was observed in each cell line, as analyzed by either or both mRNA and protein expression. Stimulation of MO7 cells with hematopoietic growth factors increased the expression of GAP as well as the levels of active GTP-bound p21ras. Stimulation of GAP expression was inhibited upon GAP antisense treatment. These data indicate that p120 GAP is involved in human normal and leukemic hemopoiesis and strongly suggest that GAP is not only a p21ras inhibitor (signal terminator), but also a positive signal transducer.

The p210bcr/abl tyrosine kinase appears to be responsible for initiating and maintaining the leukemic phenotype in chronic myelogenous leukemia (CML) patients. p21ras-p120GAP interactions play a central role in transducing mitogenic signals. Therefore, we investigated whether p21ras and p120GAP are regulated by p210bcr/abl, and whether this activation is functionally significant for CML cell proliferation. We report that transient expression of p210bcr/abl in fibroblast-like cells induces simultaneous activation of p21ras and inhibition of GTPase-promoting activity of p120GAP, and confirm these data showing that downregulation of p210bcr/abl expression in CML cells with bcr/abl antisense oligodeoxynucleotides induces both inhibition of p21ras activation and stimulation of GTPase-promoting activity of p120GAP. Tyrosine phosphorylation of two p120GAP-associated proteins, p190 and p62, which may affect p120GAP activity, also depends on p210bcr/abl tyrosine kinase expression. Direct dependence of these effects on the kinase activity is proven in experiments in which expression of c-MYB protein in fibroblast-like cells or downregulation of c-MYB expression resulting in analogous inhibition of CML cell proliferation does not result in the same changes. Use of specific antisense oligodeoxynucleotides to downregulate p21ras expression revealed a requirement for functional p21ras in the proliferation of Philadelphia chromosome-positive CML primary cells. Thus, the p210bcr/abl-dependent regulation of p120GAP activity is responsible, in part, for the maintenance of p21ras in the active GTP-bound form, a crucial requirement for CML cell proliferation.

We have identified in a human cDNA library a clone (hp2F1) whose cognate RNA is growth-regulated. The insert has been sequenced and the nucleotide sequence shows a strong homology to the nucleotide sequences of the ADP/ATP carrier cDNA and gene, respectively, isolated from Neurospora crassa and Saccharomyces cerevisiae. The putative amino acid sequence of hp2F1 shows an 87% homology to the amino acid sequence of the ADP/ATP carrier from beef heart mitochondria. We conclude that the insert of hp2F1 contains the full coding sequence of a human ADP/ATP carrier. The steady-state RNA levels of the ADP/ATP carrier are growth-regulated. They increase when quiescent cells are stimulated by serum, platelet-derived growth factor, or epidermal growth factor, but not by platelet-poor plasma or insulin. RNA levels of the ADP/ATP carrier decrease instead when growing HL-60 cells are induced to differentiate by either phorbol esters or retinoic acid.

To provide insight into the mechanisms by which c-myb regulates hematopoiesis, we analyzed the expression of markers for multiple hematopoietic lineages in differentiating parental embryonic stem (ES) cells and in ES cells transfected with c-myb or with a mutant c-myb deficient in DNA binding and assessed the ability of these cells to undergo hematopoietic commitment and colony formation. Undifferentiated ES cells transfected with intact c-myb, but not cells transfected with mutant c-myb, expressed CD34, c-kit, GATA1, and flt3 mRNA as well as surface CD34, c-kit, and flt3 product. In contrast, the kinetics of GATA-2 mRNA expression was identical in parental and Myb-transfected ES cells. Transient expression assays suggested transactivation of gene expression dependent on interaction with Myb binding sites in the CD34 and GATA1 5′ flanking regions. Undifferentiated parental and c-myb mutant-transfected ES cells were not clonogenic, whereas c-myb transfectants formed erythromyeloid colonies in methylcellulose cultures in the absence of added hematopoietic growth factors and, at higher frequency, in the presence of kit and flt-3 ligands. Colony formation was suppressed by treatment with antisense oligodeoxynucleotides specifically downregulating c-kit and flt-3 expression. These findings indicate that c-myb regulates hematopoietic commitment and progenitor cell proliferation and differentiation through the activation of certain genes that define the stem/progenitor cell compartment.

The c-myb proto-oncogene encodes a nuclear protein involved in the regulation of cell proliferation, differentiation, and development. Myb protein contains a DNA binding and a transactivating domain thought to mediate its biologic properties. The DNA binding domain consists of three repeats (R1, R2, and R3), each containing a highly conserved motif of tryptophan residues. A c-myb mutant (DR1-myb) lacking the last 46 amino acids of R1 and 23 amino terminal residues of R2, a region homologous to the ADA-2 yeast transcriptional adaptor, lost DNA binding ability, but remained able to transactivate the human heat-shock promoter. Transfection of murine 32D and murine erythroleukemia (MEL) cell lines with DR1-myb caused inhibition of cellular differentiation induced by granulocyte colony-stimulating factor (G-CSF) and dimethyl sulfoxide (DMSO), respectively. A second c-myb mutant (D-ADA2-myb) lacking the first 23 amino acids of R2, also lost DNA binding and transactivation activity, but did not inhibit DMSO-induced differentiation of MEL transfected cells. These findings suggest that deletion of R1 activates a DNA binding-independent mechanism of c-myb function, which may involve interaction of Myb with cellular factors.

To determine whether N-ras expression is required at specific stages of the process of in vitro normal human hematopoiesis, adherent- and T lymphocyte-depleted mononuclear marrow cells (A-T-MNC) or highly purified progenitors (CD34+ cells) were cultured in semisolid medium, under conditions that favor the growth of specific progenitor cell types, after exposure to N-ras sense and antisense oligodeoxynucleotides. N-ras antisense, but not sense, oligodeoxynucleotide treatment of A-T-MNC and CD34+ cells resulted in a significantly decreased number of granulocyte/macrophage colony-forming units (CFU-GM) induced by interleukin 3 (IL-3) or granulocyte/macrophage colony-stimulating factor (GM-CSF) and of macrophage colonies (CFU-M) induced by M-CSF, but not of granulocytic colonies induced with G-CSF or IL-5. However, the same treatment significantly inhibited colony formation induced by each of the above factors in combination with IL-3. Megakaryocytic colony (CFU-Meg) formation from A-T-MNC or CD34+ cells in the presence of IL-6 + IL-3 + erythropoietin (Epo) was also markedly decreased after antisense oligodeoxynucleotide treatment. Erythroid colonies derived from A-T-MNC in the presence of Epo (CFU-E) were not inhibited upon antisense treatment, whereas those arising from A-T-MNC or CD34+ cells in the presence of IL-3 + Epo (BFU-E) were markedly affected. These results are consistent with the hypothesis that distinct signal transduction pathways, involving N-ras or not, are activated by different growth factors in different hematopoietic progenitor cells.

The c-myb protooncogene plays a major role in regulating the process of in vitro and in vivo hematopoiesis via its activity as transcriptional regulator in hematopoietic progenitor cells. Since the bone marrow microenvironment appears to regulate in vivo hematopoiesis by maintaining the growth of multipotent progenitors via secretion of specific cytokines, we asked whether c-myb is also required for the proliferation of and/or cytokine production by stromal cells that generate fibroblast-like colonies (fibroblast colony-forming units [CFU-F]). Using the reverse transcriptase polymerase chain reaction technique, we detected low levels of c-myb mRNA transcripts in human normal bone marrow fibroblasts. Treatment of these cells with c-myb antisense oligodeoxynucleotides caused downregulation of c-myb expression, decreased in the number of marrow CFU-F colonies (approximately 54% inhibition) and in the cell number within residual colonies (approximately 80%), and downregulation of granulocyte/macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) mRNA expression. Transfection of T98G glioblastoma cells, in which expression of c-myb, GM-CSF, and SCF mRNAs is undetectable or barely detectable, with a plasmid containing a full-length c-myb cDNA under the control of the SV40 promoter induced the expression of biologically active SCF and GM-CSF in these cells. Regulation of GM-CSF expression by c-myb was due in part to transactivation of the GM-CSF promoter. These results indicate that, in addition to regulating hematopoietic cell proliferation, c-myb is also required for proliferation of and cytokines synthesis by bone marrow fibroblasts.

We have studied the expression of cell-cycle genes specific to the G1 (2A9, 2F1, 4F1, c-myc) and S (histone H3) phases of the cell cycle in normal and malignant human myeloid cycling cells. The levels of expression were determined by measuring the amounts of specific RNA in blot hybridization assays. Levels of expression of the G1 genes were compared to the level of expression of the S-phase-specific H3 gene. This method can distinguish whether an increased expression of G1 genes is truly due to deregulation or simply reflects an increase in the fraction of proliferating cells. In a normal asynchronous system provided by the bone marrow cells of three normal donors, the expressions of the four G1-specific genes 2A9, 2F1, 4F1, and c-myc, and of the S-phase-specific gene H3 were in ratios that differed little from one individual to another. In the total RNA of eight patients in the chronic phase of chronic myelogenous leukemia, a high level of expression of G1 cell-cycle genes was paralleled by a high level of expression of the S-phase H3 gene, simply reflecting an increase in the fraction of proliferating cells. In patients with acute myelogenous leukemia (AML), the RNA levels of 2F1 and 4F1 paralleled the expression of H3-i.e., the ratios of expression 2F1/H3 and 4F1/H3 were the same as in normal bone marrow cells. However, in 9 of 10 patients with AML we found that the expression of c-myc was elevated with respect to H3 expression. The expression of 2A9 (with respect to H3) was also elevated in some of these AML patients. Two important conclusions can be drawn from these findings: increased levels of a G1-specific RNA in a tumor may not indicate overexpression of that gene but may instead simply reflect the fraction of proliferating cells; and in some patients with AML, however, the expression of certain G1 genes is truly deregulated and might contribute to the impairment of proliferative control that is associated with this phenotype.

The transcription factors c-myb and GATA-2 are both required for blood cell development in vivo and in vitro. However, very little is known on their mechanism(s) of action and whether they impact on complementary or overlapping pathways of hematopoietic proliferation and differentiation. We report here that embryonic stem (ES) cells transfected with c-myb or GATA-2 cDNAs, individually or in combination, underwent hematopoietic commitment and differentiation in the absence of added hematopoietic growth factors but that stimulation with c-kit and flt-3 ligands enhanced colony formation only in the c-myb transfectants. This enhancement correlated with c-kit and flt-3 surface receptor up-regulation in c-myb-(but not GATA-2-) transfected ES cells. Transfection of ES cells with either a c-myb or a GATA-2 antisense construct abrogated erythromyeloid colony-forming ability in methyl cellulose; however, introduction of a full-length GATA-2 or c-myb cDNA, respectively, rescued the hematopoiesis-deficient phenotype, although only c-myb-rescued ES cells expressed c-kit and flt-3 surface receptors and formed increased numbers of hematopoietic colonies upon stimulation with the cognate ligands. These results are in agreement with previous studies indicating a fundamental role of c-myb and GATA-2 in hematopoiesis. Of greater importance, our studies suggest that GATA-2 and c-myb exert their roles in hematopoietic gene regulation through distinct mechanisms of action in nonoverlapping pathways.

Calcyclin was originally defined as a cDNA clone (2A9) whose cognate RNA is growth-regulated and whose sequence shows strong similarities to the sequences of the S-100 protein, a calcium-binding protein, as well as to a subunit of the major cellular substrate for tyrosine kinase. Using the full-length cDNA, we have now isolated from a human genomic library several phages containing calcyclin sequences. One of the phages, ch. 28-10, contains the entire calcyclin gene, plus extensive flanking sequences. The calcyclin gene is a unique copy gene and has 3 exons. The 5′ flanking sequence has been characterized, both structurally and functionally. Besides a TATA box, it contains, in the region proximate to the cap site, GC boxes and a sequence with a strong homology to the enhancer core of the SV40 promoter. Other enhancer-like elements are found scattered in both the 5′ and 3′ flanking regions. The proximate 5′ flanking region is very active in driving the transient expression of linked reporters in transfection experiments. Finally, the calcyclin gene has been localized to the long arm of human chromosome 1, near the ski oncogene.

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.

The nuclear proto-oncogene c-myb is preferentially expressed in lymphohematopoietic cells, in which it plays an important role in the processes of differentiation and proliferation. The mechanism(s) that regulates c-myb expression is not fully understood, although in mouse cells a regulatory mechanism involves a transcriptional block in the first intron. To analyze the contribution of the 5' flanking sequences in regulating the expression of the human c-myb gene, we isolated a genomic clone containing extensive 5' flanking sequences, the first exon, and a large portion of the first intron. Sequence analysis of a subcloned 1.3-kb BamHI insert corresponding to 687 nucleotides of the 5' flanking sequence, the entire first exon, and 300 nucleotides of the first intron revealed the presence of closely spaced putative Myb binding sites within a segment extending from nucleotides -616 to -575 upstream from the cap site. A 165-bp segment containing these putative Myb binding sites was linked to a human thymidine kinase (TK) cDNA driven by a low-activity proliferating cell nuclear antigen promoter and cotransfected into TK- ts13 cells with a plasmid in which a full-length human c-myb cDNA is driven by the early simian virus 40 promoter; Myb inducibility of TK mRNA expression was observed both in transient expression assays and in stable transformants. The highest level of inducibility was detected when the 165-bp fragment was placed 138 bp upstream of the proliferating cell nuclear antigen promoter-TK cDNA reporter unit or 3' of the TK cDNA. Mutation of the putative Myb binding sites greatly reduced c-myb transactivation of TK mRNA expression and specifically reduced the binding of in vitro-translated Myb protein at those sites. Finally, c-myb transactivated TK mRNA expression driven by a segment of the authentic c-myb 5' flanking region containing the Myb binding sites. These data suggest that human c-myb maintains high levels of Myb protein in cells that require this gene product for proliferation and/or differentiation by an autoregulatory mechanism involving Myb binding sites in the 5' flanking region.

The CD34 antigen defines a subset of hematopoietic progenitor cells with self-renewal capacity and the ability to reconstitute hematopoiesis in irradiated primates and marrow-ablated humans, but its function remains unknown. The c-myb protooncogene plays a fundamental role in hematopoiesis, most likely via its transcriptional regulator function. We report that c-myb protein transactivates the CD34 promoter via specific interaction with multiple Myb binding sites in the 5' flanking region of the gene and induces expression of the endogenous CD34 mRNA in rodent fibroblasts. Also, constitutive expression of c-myb in CD34-negative human glioblastoma cells induces expression of CD34 mRNA and synthesis of the surface membrane antigen. These data directly demonstrate that c-myb regulates the expression of the hematopoietic stem cell antigen CD34 and raise the possibility that c-myb regulates hematopoiesis inducing a cascade of differentiation-related events.

We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth. A greater inhibition of cell proliferation in vitro and a higher increase of tumour growth inhibition and growth delay in vivo were obtained with the combination of (S)ODNs and CDDP than when the two agents were administered separately. This comparative study, using the same tumour cell line in vitro and in vivo, suggests that c-myb antisense (S)ODNs might be useful in the therapy of colon cancer in combination with antineoplastic drugs.

The B-myb gene belongs to a family of transcription factors that also includes A-myb and c-myb. B-myb is expressed in many cell types including human neuroblastoma cells. Here we demonstrate that B-myb expression is down-regulated during retinoic acid-induced neural and glial differentiation of neuroblastoma cells. This modulation is an early event, is maintained at late times of induction, and is in part regulated at the transcriptional level. Constitutive expression of B-myb prevents retinoic acid-induced neural differentiation as reflected by morphological features and the expression of (or lack of) biochemical markers associated with the undifferentiated phenotype. Furthermore, the expression of antisense B-myb transcripts does not allow the rescue of viable cells, suggesting an important role for B-myb in the survival of neuroblastoma cells. These results indicate that B-myb plays a functional role in the differentiative potential of neuroblastoma cells, raising the possibility that this gene is one of the nuclear regulators in the cascade of events leading to cellular differentiation.

The role of the KIT protooncogene in human hematopoiesis is uncertain. Therefore, we examined KIT mRNA expression in normal human bone marrow mononuclear cells (MNC) and used antisense oligodeoxynucleotides (oligomers) to disrupt KIT function. KIT mRNA was detected with certainty only in growth factor-stimulated MNC. Expression was essentially abrogated by making MNC quiescent or by inhibiting myb gene function. Oligomers blocked KIT mRNA expression in a dose-response and sequence-specific manner, thereby allowing functional examination of the KIT receptor. In experiments with either partially purified or CD34(+)-enriched MNC, neither granulocyte nor megakaryocyte colony formation was inhibited by oligomer exposure. In contrast, KIT antisense oligomers inhibited interleukin 3/erythropoietin-driven erythroid colony formation approximately 70% and "stem cell factor"/erythropoietin-driven colony formation 100%. The presence of erythroid progenitor cell subsets with differential requirements for KIT function is therefore suggested. Growth of hematopoietic colonies from chronic myeloid leukemia and polycythemia vera patients was also inhibited, while acute leukemia colony growth appeared less sensitive to KIT deprivation. These results suggest that KIT plays a predominant role in normal erythropoiesis but may be important in regulating some types of malignant hematopoietic cell growth as well. They also suggest that KIT expression is linked to cell metabolic activity and that its expression may be regulated by or coregulated with MYB.

Three cDNA clones isolated from Syrian hamster cells (p4F1, p2F1, and p2A9) contain sequences that are preferentially expressed in the G1 phase of the cell cycle. The expression of these sequences was investigated in human peripheral blood cells from normal individuals and from patients with leukemia. The expression of p4F1 and p2F1 is clearly dependent on the cell cycle in peripheral blood mononuclear cells stimulated to proliferate with phytohemagglutinin; the p2A9 sequences cannot be clearly detected in human lymphocytes but are expressed in a cell-cycle-dependent manner in human diploid fibroblasts (WI-38). These genes also show different levels of expression in lymphoid and myeloid leukemias. The highest level of expression for p2A9 is found in patients with chronic myelogenous leukemia, and the lowest in patients with chronic lymphocytic leukemia. For p2F1 and p4F1, the highest levels of expression are found in chronic and acute myelogenous leukemia. At least two other cell-cycle genes are not expressed at detectable levels in human leukemias. These findings suggest that the activation of cell-division-cycle genes might contribute, like cellular oncogenes, to the phenotype of human malignancies and that, perhaps, new oncogenes could be found by identifying and isolating genes whose expression is dependent on the cell cycle.

The BCR/ABL oncogenic tyrosine kinase is responsible for initiating and maintaining the leukemic phenotype of Philadelphia chromosome (Ph1)- positive cells. Phosphatidylinositol-3 (PI-3) kinase is known to interact with and be activated by receptor and nonreceptor tyrosine kinases. We investigated whether PI-3 kinase associates with and/or is regulated by BCR/ABL, whether this interaction is functionally significant for Ph1 cell proliferation, and, if so, whether inhibition of PI-3 kinase activity can be exploited to eliminate Ph1-positive cells from bone marrow. We show that the p85 alpha subunit of PI-3 kinase associates with BCR/ABL and that transient expression of BCR/ABL in fibroblasts and down-regulation of BCR/ABL expression using antisense oligodeoxynucleotides (ODNs) in Ph1 cells activates and inhibits, respectively, PI-3 kinase enzymatic activity. The use of specific ODNs or antisense constructs to downregulate p85 alpha expression showed a requirement for p85 alpha subunit in the proliferation of BCR/ABL-dependent cell lines and chronic myelogenous leukemia (CML) primary cells. Similarly, wortmannin, a specific inhibitor of the enzymatic activity of the p110 subunit of PI-3 kinase, inhibited growth of these cells. The growth of normal bone marrow and erythromyeloid, but not megakaryocyte, progenitors was inhibited by p85 alpha antisense [S]ODNs, but wortmannin, at the concentrations tested, did not affect normal hematopoiesis. The proliferation of two BCR/ABL- and growth factor-independent cell lines was not affected by downregulation of the expression of the p85 alpha subunit or inhibition of p110 enzymatic activity, confirming the specificity of the observed effects on Ph1 cells. Thus, PI-3 kinase is one of the downstream effectors of BCR/ABL tyrosine kinase in CML cells. Moreover, reverse transcriptase-polymerase chain reaction performed on single colonies to detect BCR-ABL transcripts showed that wortmannin was able to eliminate selectively CML-blast crisis cells from a mixture of normal bone marrow and Ph1 cells.

When injected into SCID mice, the Philadelphia chromosome-positive chronic myeloid leukemia-blast crisis cell line BV173 induces a disease process closely resembling that seen in leukemia patients. At 1 and 3 weeks after injection of 10(6) BV173 cells, CD10+ cells were detected in the bone marrow of the mice, leukemic colonies grew from bone marrow and spleen cell suspensions, and BCR-ABL transcripts were detectable in bone marrow, spleen, peripheral blood, liver, and lungs. Systemic treatment of the leukemic mice with a 26-mer BCR-ABL antisense oligodeoxynucleotide (1 mg/day for 9 days) induced disappearance of CD10+ and clonogenic leukemic cells and a marked decrease in BCR-ABL mRNA in mouse tissues. Untreated mice or mice treated with a BCR-ABL sense oligodeoxynucleotide or a 6-base-mismatched antisense oligodeoxynucleotide oligodeoxynucleotide were dead 8-13 weeks after leukemia cell injection; in marked contrast, mice treated with BCR-ABL antisense oligodeoxynucleotide died of leukemia 18-23 weeks after injection of leukemic cells. These findings provide evidence for the in vivo effectiveness of an anticancer therapy based on antisense oligodeoxynucleotides targeting a tumor-specific gene.