Biomedical subjects

D Perrotti

Publications and source records attributed to D Perrotti.

26 records · Page 2Linked to original sources

PubMed1996

Blastic transformation of p53-deficient bone marrow cells by p210bcr/abl tyrosine kinase.

Blastic transformation of chronic myelogenous leukemia (CML) is characterized by the presence of nonrandom, secondary genetic abnormalities in the majority of Philadelphia1 clones, and loss of p53 tumor suppressor gene function is a consistent finding in 25-30% of CML blast crisis patients. To test whether the functional loss of p53 plays a direct role in the transition of chronic phase to blast crisis, bone marrow cells from p53+/+ or p53-/- mice were infected with a retrovirus carrying either the wild-type BCR/ABL or the inactive kinase-deficient mutant, and were assessed for colony-forming ability. Infection of p53-/- marrow cells with wild-type BCR/ABL, but not with the kinase-deficient mutant, enhanced formation of hematopoietic colonies and induced growth factor independence at high frequency, as compared with p53+/+ marrow cells. These effects were suppressed when p53-/- marrow cells were coinfected with BCR/ ABL and wild-type p53. p53-deficient BCR/ABL-infected marrow cells had a proliferative advantage, as reflected by an increase in the fraction of S+G2 phase cells and a decrease in the number of apoptotic cells. Immunophenotyping and morphological analysis revealed that BCR/ABL-positive p53-/- cells were much less differentiated than their BCR/ABL-positive p53+/+ counterparts. Injection of immunodeficient mice with BCR/ABL-positive p53-/- cells produced a transplantable, highly aggressive, poorly differentiated acute myelogenous leukemia. In marked contrast, the disease process in mice injected with BCR/ABL-positive p53+/+ marrow cells was characterized by cell infiltrates with a more differentiated phenotype and was significantly retarded, as indicated by a much longer survival of leukemic mice. Together, these findings directly demonstrate that loss of p53 function plays an important role in blast transformation in CML.

Animals↗

PubMed1996

A cell proliferation-dependent multiprotein complex NC-3A positively regulates the CD34 promoter via a TCATTT-containing element.

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.

Animals↗

PubMed1995

C-RAF-1 serine/threonine kinase is required in BCR/ABL-dependent and normal hematopoiesis.

BCR/ABL oncogenic tyrosine kinase is responsible for initiating and maintaining the leukeic phenotype of Philadelphia chromosome-positive cells. c-RAF-1 serine/threonine kinase is known to be activated by receptor and nonreceptor tyrosine kinases. To determine whether c-RAF-1 plays a role in the growth of BCR/ABL-dependent cells, we examined whether c-RAF-1 associates with and/or is regulated by BCR/ABL and, if so, whether this interaction is functionally significant for BCR/ABL-dependent growth of chronic myelogenous leukemia cells and for growth factor-dependent proliferation of normal bone marrow cells. We show that c-RAF-1 enzymatic activity is regulated by BCR/ABL, although the protein does not associate with BCR/ABL. Downregulation of c-RAF-1 expression with antisense oligodeoxynucleotides or cDNA constructs, and inhibition of c-RAF-1 activity by its dominant negative mutants, inhibited both BCR/ABL-dependent growth of chronic myelogenous leukemia cells and growth factor-dependent proliferation of normal hematopoietic progenitors and the MO7 cell line without affecting the BCR/ABL-and growth factor-independent proliferation of HL-60 cells. These results indicate that c-RAF-1 plays an important role in Philadelphia chromosome-positive and normal hematopoiesis.

Base Sequence↗

PubMed1995

Structure and growth-dependent regulation of the human cyclin B1 promoter.

As a step toward defining the signals important for the regulation of cyclin B1 expression, we cloned, sequenced, and partially characterized a 1012-bp genomic fragment encompassing the human cyclin B1 promoter. By transient expression experiments, we found that promoter activity resides within a -150/+182-bp DNA fragment. The activity of this promoter fragment was high in asynchronous NIH-3T3 cells, but dramatically decreased in quiescent cells. Time-course experiments, using stable transfectants expressing the CAT gene under the control of this fragment, were performed after releasing the cells from serum starvation. The results showed that the promoter becomes active at the end of the S phase and its activity increases during the cell cycle. Similar experiments performed with a shorter promoter region (-58/+182) showed that this 5' deletion mutant is active throughout the cell cycle. In good agreement with promoter activity, Northern analysis indicated that the endogenous gene is negatively regulated in quiescent murine NIH-3T3 cells. The data presented here demonstrate that in NIH-3T3 cells the cyclin B1 promoter is growth regulated, and important regulatory elements must exist in the region spanning -150 to -58 bp.

3T3 Cells↗

PubMed1995

Overexpression of the zinc finger protein MZF1 inhibits hematopoietic development from embryonic stem cells: correlation with negative regulation of CD34 and c-myb promoter activity.

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.

Amino Acid Sequence↗

PubMed1992

Inhibition of leukemia cell proliferation by receptor-mediated uptake of c-myb antisense oligodeoxynucleotides.

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.

Antineoplastic Agents↗

PubMed1991

Differential expression of transforming growth factor-beta 1 gene in 3LL metastatic variants.

In vitro and in vivo metastatic variants derived from Lewis lung carcinoma (3LL) were examined for the level of the expression of several growth-regulated genes, oncogenes, and transforming growth factor (TGF) genes. To determine whether the proliferative advantage of metastatic cells is due to an increased growth fraction of the cell population or to a deregulated expression of some growth-regulated genes, the mRNA levels of the S-phase-specific H3 histone gene were compared with that of some cell cycle-related genes (vimentin, calcyclin, c-myc, and p53) and oncogenes (Ki-ras, Ha-ras, c-sis, c-src, c-fes, and c-erb). In addition, to evaluate whether an autocrine pattern of cell proliferation is responsible for the proliferative advantage of metastatic cells, the level of the expression of TGF genes (alpha and beta 1) was studied. Northern blot analysis demonstrated that in 3LL metastatic variants the expression of TGF-alpha as well as the expression of all growth-regulated genes and oncogenes studied are similar. Only the TGF-beta 1 gene is expressed at higher levels in highly metastatic 3LL variants maintained either in vitro or in vivo. Data suggest that the proliferative advantage of 3LL metastatic cells is not due to a deregulated expression of some growth-regulated genes and oncogenes, but more likely is acquired through the expression of genes which might interfere with the ability of the tumor cells to escape hostile microenvironmental conditions.

Animals↗

PubMedDate not supplied

Metastatic phenotype: growth factor dependence and integrin expression.

Homogeneous subpopulations, which are endowed with low or high metastatic potential, were selected from Lewis lung carcinoma (3LL) in an attempt to correlate metastatic phenotype with specific properties of tumor cells. Since the growth of malignant cells at secondary sites could depend on their ability to respond to microenvironments, the growth factor dependence of 3LL variants has been studied. The ability of variant lines to grow in monolayer and in soft agar cultures, either in the presence or absence of different growth factors or serum, was analyzed and correlated with their metastatic potential. The reported results demonstrate that tumor cells expressing higher metastatic potential also exhibit higher capacity to grow and proliferate in all the culture conditions tested, independently of the addition of exogenous growth factors or serum. Moreover, since highly metastasizing cells express a significant amount of TGF-beta 1 mRNA, a pattern of autocrine growth is postulated for 3LL metastatic cells. One relevant aspect of the phenotype of transformed cells is their reduced adhesion to solid substrates; this phenomenon is thought to reflect the invasive and metastatic potential of tumor cells. Since the adhesion of the cells to substrata is mediated by molecules of the extracellular matrix, the expression of extracellular matrix receptors (integrins) was studied on 3LL metastatic variants. In particular, through immunochemical and biochemical studies we investigated the expression of the laminin receptor(alpha 6/beta 1) and of a novel receptor (integrin: alpha 6/beta 4), of unknown function. The receptors were quantitated on the cell surface of 3LL variants by the use of specific monoclonal antibodies which recognize, respectively, different epitopes of alpha 6, beta 4 or beta 1 subunits. Results demonstrate that the novel integrin alpha 6/beta 4, is specifically expressed in highly metastasizing 3LL cells, whereas the laminin receptor alpha 6/beta 1 is expressed in all 3LL variants. In conclusion, data presented demonstrate that 3LL cells endowed with higher metastatic potential are more independent of the microenvironmental conditions in that they possess a higher autocrine capacity than the lower metastasizing ones, and could acquire higher capacity to invade through the expression on their cell surface of specific receptors for cell adhesion (the novel integrin, defined as alpha 6/beta 4).

Animals↗