Pharmacological strategies for overcoming multidrug resistance.

Multidrug resistance (MDR) is a major obstacle to the effective treatment of cancer. One of the underlying mechanisms of MDR is cellular overproduction of P-glycoprotein (P-gp) which acts as an efflux pump for various anticancer drugs. P-gp is encoded by the MDR1 gene and its overexpression in cancer cells has become a therapeutic target for circumventing multidrug resistance. A potential strategy is to co-administer efflux pump inhibitors, although such reversal agents might actually increase the side effects of chemotherapy by blocking physiological anticancer drug efflux from normal cells. Although many efforts to overcome MDR have been made using first and second generation reversal agents comprising drugs already in current clinical use for other indications (e.g. verapamil, cyclosporine A, quinidine) or analogues of the first-generation drugs (e.g. dexverapamil, valspodar, cinchonine), few significant advances have been made. Clinical trials with third generation modulators (e.g. biricodar, zosuquidar, and laniquidar) specifically developed for MDR reversal are ongoing. The results however are not encouraging and it may be that the perfect reverser does not exist. Other approaches to multidrug resistance reversal have also been considered: encapsulation of anthracyclines in liposomes or other carriers which deliver these drugs selectively to tumor tissues, the use of P-gp targeted antibodies such as UIC2 or the use of antisense strategies targeting the MDR1 messenger RNA. More recently, the development of transcriptional regulators appears promising. Also anticancer drugs that belong structurally to classes of drugs extruded from cells by P-gp but that are not substrates of this drug transporter may act as potent inhibitors of MDR tumors (e.g. epothilones, second generation taxanes). Taking advantage of MDR has also been studied. Bone marrow suppression, one of the major side effects of cancer chemotherapy, can compromise the potential of curative and palliative chemotherapy. It is conceivable that drug resistance gene transfer into bone marrow stem cells may be able to reduce or abolish chemotherapy-induced myelosuppression and facilitate the use of high dose chemotherapy. Clinical trials of retroviral vectors containing drug resistance genes have established that the approach is safe and are now being designed to address the therapeutically relevant issues.

Regulation of mouse p45 NF-E2 transcription by an erythroid-specific GATA-dependent intronic alternative promoter.

The erythroid-enriched transcription factor NF-E2 is composed of two subunits, p45 and p18, the former of which is mainly expressed in the hematopoietic system. We have isolated and characterized the mouse p45 NF-E2 gene; we show here that, similar to the human gene, the mouse gene has two alternative promoters, which are differentially active during development and in different hematopoietic cells. Transcripts from the distal promoter are present in both erythroid and myeloid cells; however, transcripts from an alternative proximal 1b promoter, lying in the first intron, are abundant in erythroid cells, but barely detectable in myeloid cells. During development, both transcripts are detectable in yolk sac, fetal liver, and bone marrow. Transfection experiments show that proximal promoter 1b has a strong activity in erythroid cells, which is completely dependent on the integrity of a palindromic GATA-1 binding site. In contrast, the distal promoter 1a is not active in this assay. When the promoter 1b is placed 3' to the promoter 1a and reporter gene, in an arrangement that resembles the natural one, it acts as an enhancer to stimulate the activity of the upstream promoter la.

Expression in hematopoietic cells of GATA-1 transcripts from the alternative "testis" promoter during development and cell differentiation.

GATA-1 is a transcription factor expressed both in the hematopoietic system and in the Sertoli cells of the testis, and is essential for correct erythropoiesis. Hematopoietic and Sertoli cells transcribe GATA-1 from two different promoters: the proximal (erythroid) is active in hematopoietic cells; the distal (testis) is active in Sertoli cells. We investigated by RT-PCR the possibility that GATA-1 might be transcribed from the testis promoter also in hematopoietic cells. Testis promoter-derived transcripts are present at low levels in vivo at all stages of hematopoietic development. Purified multipotent progenitors, fractionated into populations expressing low or high levels of GATA-1, do not contain any "testis" transcripts. However, when grown in vitro, they rapidly express GATA-1 from the testis promoter in the presence of Erythropoietin (Epo) but not in that of other growth factors. This result reflects an Epo-dependent differentiation event, rather than a direct effect of Epo. Indeed, immortalized progenitor cell lines which respond to both Epo and SCF, continue to express testis-derived transcripts when switched from Epo to SCF.

Erythroid-specific activation of the distal (testis) promoter of GATA1 during differentiation of purified normal murine hematopoietic stem cells.

To understand the molecular mechanisms of erythroid differentiation, we analyzed by semiquantitative RT-PCR the expression of the transcription factor GATA1, the erythropoietin receptor (EpoR), and erythroid (beta-globin) differentiation markers in purified hematopoietic stem cells (HSCs) after in-vitro-induced differentiation. Whether GATA1 transcription was from the proximal (with respect to the AUG, also known as erythroid) or the distal (also known as testis) promoter was analyzed as well. Low-density marrow cells which bind to wheat germ agglutinin, but not to the antibody 15.1.1, and which either do or do not retain the dye rhodamine-123 (Rho-bright and Rho-dull, respectively), were purified. Rho-dull, but not Rho-bright cells permanently reconstitute lymphomyelopoiesis in W/Wv and severe-combined-immunodeficiency mice and, therefore, contain HSCs. Both Rho-dull and Rho-bright cells give rise to progenitor and differentiated cells (peak values at days 15 and 5, respectively) in liquid culture. Multilineage, erythroid-restricted or myeloid-restricted differentiation is observed when the cultures are stimulated with stem cell factor (SCF) + interleukin (IL)-3, SCF + IL-3 + Epo, or SCF + IL-3 + granulocyte-colony-stimulating factor, respectively. Rho-dull cells have barely detectable reconstitution potential at day 5 of culture. None of the genes examined were expressed in purified Rho-bright or Rho-dull cells. The only exception was GATA1 which was expressed at maximal levels in Rho-bright cells at the onset of culture. Rho-dull cells did not express GATA1 before day 3 of culture (maximal expression at days 10-15). Activation of GATA1 and EpoR was observed in all growth of mRNA for the two genes expressed by the cells. In contrast, beta-globin mRNA was detected only in the presence of Epo. The transcription of GATA1 was exclusively from the proximal promoter in the absence of Epo but both proximal and distal transcripts were observed in its presence. Maximum transcription from the distal promoter (approximately equal to 0.2% of total GATA1 mRNA) coincided with maximal globin mRNA levels (day 5 or day 15 for Rho-bright and Rho-dull cells, respectively). These results indicate that GATA1 is activated at the transition point between HSCs and pluripotent progenitor cells and erythroid-specific GATA1 regulation involves activation of the distal GATA1 promoter.

Immortalization of multipotent growth-factor dependent hemopoietic progenitors from mice transgenic for GATA-1 driven SV40 tsA58 gene.

The transcription factor GATA-1 is required for the normal development of erythroid cells. GATA-1 is also expressed in other hemopoietic cells, suggesting that it might be initially activated in a multipotent progenitor. To immortalize GATA-1-expressing progenitors, we generated mice transgenic for a thermosensitive SV40 T gene, driven by the GATA-1 promoter-enhancer. Immortalized marrow cells grow in culture at 32 degrees C but not at 38 degrees C, and are dependent on erythropoietin (Epo) or interleukin 3 (IL-3). Epo dependent cells express hemoglobin, high levels of GATA-1, GATA-2 and NF-E2 p45 mRNAs, and are positive for stem cell antigen 2 (Sca-2) and the early myeloid marker ER-MP12. IL-3 dependent cells can be derived from Epo dependent lines, and are hemoglobin-, Sca-2- and ER-MP12-negative, have low GATA-1 and NF-E2 p45 mRNA levels, and express myeloid markers Mac-1, F4/80 and Gr-1. Brief treatment of Epo dependent cells with myeloid growth factors (plus Epo) leads to the induction of Mac-1, F4/80 and Gr-1, concomitant with the disappearance from most cells of Sca-2, ER-MP12 and GATA-1 driven T antigen nuclear expression. Thus, the immortalized Epo dependent cells have the property of a progenitor capable of differentiation towards either the erythroid or myeloid lineages. These cells initiate transcription of a proportion of GATA-1 RNA molecules at an upstream promoter, previously known to be expressed only in testis cells.

4-Hydroxynonenal, a product of cellular lipid peroxidation, which modulates c-myc and globin gene expression in K562 erythroleukemic cells.

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. Here, we show that 4-hydroxynonenal (HNE) concentrations close to the level found in normal cells (in the range of 1 and 3 microM) can specifically induce changes in the expression of c-myc and gamma-globin mRNA in K562 cells, without inducing any toxic effects or affecting cell viability. Since we have determined that K562 cells have undetectable levels of endogenous lipid peroxidation, all these effects can be assigned to the exogenous HNE treatment. After a 1-h treatment with 1 microM HNE, c-myc mRNA levels decrease transiently during the first 4 h, rebounding later to higher levels, and normalizing to basal expression after 4 days. Run-on experiments show a transient transcriptional block 20 min after HNE treatment and subsequent posttranscriptional regulation. According to S1 mapping, mRNA changes are exerted on c-myc transcripts initiated from both the principal constitutive start sites (P1 and P2). gamma-Globin mRNA levels concomitantly increase 3- to 4-fold, but no significant changes of housekeeping gene expression are observed. On the basis of these results it appears that the restoration in human erythroleukemic K562 cells of HNE concentrations closer to the level in normal cells can modulate the expression of specific genes.

Tumorigenic activity of a rearranged c-myc gene from a human T-cell leukemia line.

The T-lymphoma cell line Hut78 contains a rearranged c-myc oncogene derived from a translocation between the long arms of chromosomes 8 and 2; the event deletes the 3' end of the gene, causing the loss of the transcribed AT-rich sequence. It has recently been shown that the mutant c-myc mRNA is several-fold more stable than normal c-myc mRNA. We have assessed the tumorigenicity of the mutant c-myc allele by transfecting this gene and its normal counterpart into NIH3T3 cells, together with a neomycin resistance gene. Following selection for G-418 resistance, the cells were injected into nude mice. Tumors containing integrated c-myc arose in animals injected with cells transfected by the mutated, but not by the normal, allele. The results suggest that this rearranged c-myc bears a tumorigenic activity not observed in other naturally occurring mutated c-myc alleles and may have directly contributed to the tumorigenic event in the Hut78 cell line.

An erythroid specific enhancer upstream to the gene encoding the cell-type specific transcription factor GATA-1.

The transcription factor GATA-1 is expressed in a subset of hemopoietic cells, where it mediates the cell-type specific expression of several genes. We have cloned the mouse and human GATA-1 genes. A region upstream to the first exon, and highly conserved between mouse and man, acts as an erythroid specific enhancer in transient assays, if linked to the GATA-1 or to the SV40 promoter. The activity of the enhancer is almost completely dependent on the integrity of a dimeric GATA-1 binding site.

Mapping the gene encoding the human erythroid transcriptional factor NFE1-GF1 to Xp11.23.

The X-linked NFE1 gene encodes an erythroid factor involved in globin gene transcription. Using a human cDNA clone encoding this factor, we show, by in situ hybridization and by analysis of human-rodent hybrid cell lines, that this gene is located in Xp11.23. In the absence of polymorphisms in the NFE1 gene, these results allow the study of the possible relationships between NFE1 mutations and X-linked hereditary persistence of fetal hemoglobin by linkage analysis with RFLP markers of the region. A female patient, hemizygous for the NFE1 locus, shows essentially normal hematological parameters.

The same nuclear proteins bind the proximal CACCC box of the human beta-globin promoter and a similar sequence in the enhancer.

Using in vitro assays, we show that nuclear proteins related to the Sp1 and GT-1 factors bind to a CACCC box sequence in the human beta-globin enhancer, adjacent to binding sites for the erythroid-specific factor NFE1 and the ubiquitous factor CP1. The same proteins are known to bind to the proximal, but not to the distal, CACCC, box in the human beta-globin promoter. A C G mutation in the promoter CACCC box, known to cause beta-thalassemia, greatly decreases protein binding to the CACCC box; the same effect is obtained when this mutation is introduced into the enhancer CACCC box.

Increased erythroid-specific expression of a mutated HPFH gamma-globin promoter requires the erythroid factor NFE-1.

The -175 T greater than C mutation in the promoter of the A gamma- or G gamma-globin gene causes a 50-100 fold increase of the expression of the respective gene in adult erythroid cells (Hereditary Persistence of Fetal Hemoglobin). We show here that this mutation increases 3-9 fold the expression of a gamma-CAT reporter plasmid transfected into the erythroid cells K562, but not that of the same plasmid in non erythroid cells. The overexpression of the mutant is abolished by the mutation of the binding site for the erythroid specific factor NFE1; inactivation of the adjacent binding site for the ubiquitous factor OTF1 does not cause overexpression of the normal gamma-globin promoter. Previous results demonstrated that the -175 mutation slightly increases the in vitro binding of NFE1 and almost abolishes that of OTF1; the present functional data indicate that altered binding of NFE1, but not of OTF1, is responsible for the observed overexpression of the mutated promoter.

DNA sequences regulating human globin gene transcription in nondeletional hereditary persistence of fetal hemoglobin.

Strong genetic evidence supports the idea that point mutations in the promoter of gamma-globin genes overexpressed in adult age [hereditary persistence of fetal hemoglobin (HPFH)] are responsible for the observed phenotype. DNA binding sites for ubiquitous and/or erythroid specific nuclear proteins correlate in location with the positions of point mutations responsible for HPFH. The analysis of the effects of one of these mutations (-175 T greater than C) on in vitro binding of nuclear proteins and on the activity of the mutated promoter in transfection assays indicates that altered binding of the erythroid-specific protein NFE-1 may be responsible for increased activity of the mutated promoter. Other HPFH mutations close to the distal CCAAT box (-117 G greater than A and 13 nucleotide deletions, -114 to -102) have complex effects on in vitro binding of nuclear proteins; their only common effect is the loss of binding of the erythroid-specific factor NFE3. If mechanisms generating the HPFH phenotype are homogeneous, NFE3 might be a negatively acting factor; alternatively, heterogeneous mechanisms might operate and HPFH might additionally be related to loss of binding to the distal CCAAT box region of either NFE1 (-117 HPFH) or of the ubiquitous CCAAT displacement protein-CDP (13 nucleotides deletion). Finally, it is also proposed that increased activity of the HPFH promoters may secondarily cause decreased expression of the delta- and beta-globin genes in cis possibly by competition between gamma- and beta-globin promoters for interaction with common regulatory elements.

The effects of HPFH mutations in the human gamma-globin promoter on binding of ubiquitous and erythroid specific nuclear factors.

Genetic evidence indicates that single point mutations in the gamma-globin promoter may be the cause of high expression of the mutated gene in the adult period (Hereditary Persistence of Fetal Hemoglobin, HPFH). Here we show that one of these mutations characterized by a T----C substitution at position -175 in a conserved octamer (ATGCAAAT) sequence, abolishes the ability of a ubiquitous octamer binding nuclear protein to bind a gamma-globin promoter fragment containing the mutated sequence; however, the ability of two erythroid specific proteins to bind the same fragment is increased three to five fold. DMS interference and binding experiments with mutated fragments indicate that the ubiquitous protein recognizes the octamer sequence, while the erythroid specific proteins B2, B3 recognize flanking nucleotides. Competition experiments indicate that protein B2 corresponds to an erythroid-specific protein known to bind to a consensus GATAG sequence present at several locations in alpha, beta and gamma-globin genes. Although the distal CCAAT box region of the gamma-globin gene shows a related sequence, an oligonucleotide including this sequence does not show any ability to bind the above mentioned erythroid protein; instead, it binds a different erythroid specific protein, in addition to a ubiquitous protein. The -117 G----A mutation also known to cause HPFH, and mapping two nucleotides upstream from the CCAAT box, greatly decreases the binding of the erythroid-specific, but not that of the ubiquitous protein, to the CCAAT box region fragment.

An erythroid specific nuclear factor binding to the proximal CACCC box of the beta-globin gene promoter.

We have used the gel retardation and DNAase I assays to investigate the binding of nuclear proteins to the human beta-globin promoter. Upon incubation with beta-globin promoter fragments containing the duplicated CACCC boxes, nuclear proteins from human erythroid cells generate complexes yielding four retarded bands in acrylamide gels; the three slowest bands are common to both erythroid and non erythroid cells. The fast band is present only in K562 erythroleukemic cells induced to differentiation and hemoglobin accumulation and in fetal and adult erythroblasts, but absent in uninduced K562 cells. Binding occurs on a short DNA region including the proximal CACCC box, and is not significantly competed by excess gamma-globin fragments containing the CACCC box; the CACCC box appears to be essential for this binding, as shown by the failure of a fragment containing a natural beta-thalassemic mutation (-87, C----G) to bind significantly to nuclear factors. These data suggest that the erythroid specific CACCC binding factor might play a role in the developmental activation of beta-globin transcription.

A frequent A gamma-hereditary persistence of fetal hemoglobin in northern Sardinia: its molecular basis and hematologic phenotype in heterozygotes and compound heterozygotes with beta-thalassemia.

A survey of hemoglobinopathies in northern Sardinia revealed a high frequency (0.3%) of carriers of a hematologic condition characterized by increased expression of fetal hemoglobin during adult life (hereditary persistence of fetal hemoglobin or HPFH). In spite of a normal hematologic phenotype, the heterozygous carriers for this condition display about 12% HbF, almost exclusively of the A gamma type; compound heterozygotes with beta-thalassemia have 20%-26% HbF and run a very mild clinical course. The sequence analysis of the cloned A gamma gene linked to the HPFH determinant revealed the presence of a G----A substitution at position -117 of the A gamma-globin gene promoter; the same mutation occurs also in Greek HPFH, although associated with different restriction polymorphisms. Another hereditary condition characterized by increased HbF (alpha 2 A gamma 2) level and a mild thalassemia phenotype in Sardinia is associated with the -196C----T substitution in the A gamma-globin gene promoter (Sardinian delta beta-thalassemia). Population studies using oligonucleotides complementary both to the -117 G----A and -196C----T mutations and the corresponding normal sequences confirm the presence of these mutations only in HPFH and delta beta-thalassemia chromosomes and exclude these changes being common DNA polymorphisms.

Sardinian G gamma-HPFH: a T----C substitution in a conserved "octamer" sequence in the G gamma-globin promoter.

A survey of hemoglobinopathies in Northern Sardinia allowed the identification of two subjects heterozygous for a new type of G gamma hereditary persistence of fetal hemoglobin (HPFH). The G gamma-globin gene from the HPFH chromosome shows the presence of a T----C substitution 175 nucleotides upstream of the CAP site, adding a new example of single-point mutations occurring in the promoter region of the gamma-globin genes and linked to HPFH phenotypes. In this case the mutation affects the 3' end nucleotide of a conserved octamer sequence known to be present in other regulatory elements of several genes.

A protein factor binding to an octamer motif in the gamma-globin promoter disappears upon induction of differentiation and hemoglobin synthesis in K562 cells.

Using the electrophoretic mobility shift assay and the footprinting technique, we studied the binding of nuclear proteins from erythroid and non erythroid human cells to the promoter region of the human gamma-globin gene. Two regions (A and B) of the promoter are bound by proteins present in uninduced K562 cells, but not in induced K562 cells nor in fetal liver erythroblasts; a protein binding to region A is also present in a variety of lymphoid and myeloid cells. Region B is centered on an octamer sequence identical to that present in immunoglobulin promoter and enhancers and other eukaryotic promoters; a B region binding protein common to K562 and other cells efficiently binds the octamer containing region of the histone H2B gene, while different B region proteins are more specific for uninduced K562 cells and the gamma-globin octamer containing fragment. The possible role of these nuclear proteins in gamma-globin gene regulation and/or cell differentiation is discussed.

Sardinian delta beta zero-thalassemia: a further example of a C to T substitution at position -196 of the A gamma globin gene promoter.

Selective overexpression (50- to 100-fold) in adult erythroid cells of either G gamma or A gamma fetal globin gene is observed in hereditary conditions known as delta beta zero-thalassemia and hereditary persistence of fetal hemoglobin (HPFH). Recently, a C----T change at position -196 of an overexpressed A gamma globin gene from an Italian HPFH was hypothesized, on the basis of indirect evidence, to represent the cause of the functional defect. We now show that the same mutation is present in a different overexpressed A gamma-globin gene from a Sardinian patient with a different syndrome (delta beta zero-thalassemia). The Sardinian A gamma globin gene differs from both the HPFH and the normal A gamma globin gene at nucleotide 1,560 in the noncoding portion of the third exon, where an A is deleted. In addition, the mutant -196 A gamma-globin gene is linked to a normal beta globin gene in HPFH, and to a beta-thalassemic gene (beta 39CAG----TAG) in delta beta zero-thalassemia. These data strengthen the suggestion that -196 mutation is causally linked to the abnormal phenotype and raise the question of whether the same or multiple mutational events are responsible for the appearance of the -196 mutation in different syndromes.