Human T-lymphotropic virus type 1 infection.

Human T-cell lymphotropic virus type-1 (HTLV-1) is aetiologically associated with adult T-cell leukaemia/lymphoma (ATL). HTLV-1 infection can also lead to various non-malignant diseases, for example, HTLV-1 associated myelopathy/tropical spastic paraparesis and HTLV-1 uveitis. HTLV-1 is endemic in southern Japan and the Caribbean. HTLV-1 infection is mainly transmitted by either breast-feeding, sexual intercourse or blood transfusions. Primary prevention of HTLV-1 in endemic areas by screening of blood and by refraining from breast-feeding have been successful. The incidence of ATL is rather low among HTLV-1 carriers (<5%). The precise mechanism of development of ATL remains unknown. It is a multiple-step process which does not require viral expression in the later stages of leukaemogenesis. Many samples have mutations of the tumour suppressor genes, p53 and/or p16(INK4A). Four subtypes of ATL have been identified, each having distinctive clinical features. Monoclonal integration of HTLV-1 proviral DNA into tumour cells is found in each of the subtypes. At present, no effective therapy for ATL exists.

Modulation of CD95 in leukemia and lymphoma cells by retinoids.

Retinoids are highly active for the treatment of acute promyelocytic leukemia, but have effects on many different types of normal and transformed cells. CD95 is a member of the TNF-receptor superfamily and can mediate apoptosis. In this study, we screened a series of 7 myeloid and 12 lymphoid cell lines for the expression of CD95 and for the induction of CD95 by retinoids. A differential response was observed: in 3/7 myeloid cell lines a significant down-regulation of CD95 happened which correlated with the induction of differentiation. A significant induction of CD95 was observed in 4/ 12 lymphoid lines (2 HHV8+ cell lines, one Burkitt line and another lymphoma line). The modulation of CD95 was both time- and dose- dependent (maximum reached after 3-5 days of culture with 10-6 M retinoid). The 3 retinoid compounds studied (all-trans retinoic acid, 9-cis retinoic acid and 13-cis retinoic acid) had comparable activity. Taken together, we describe a new mode of induction of CD95 in lymphoma cells. More work is needed to define the functional consequences of these changes in CD95 surface expression.

p53 alterations in uterine leiomyosarcomas versus leiomyomas.

Leiomyoma is the most common benign smooth muscle tumor. Although rare in other organs, it is frequent in the uterus, occurring in nearly 40% of women over 50 years of age. These benign tumors rarely undergo malignant transformation. The incidence of leiomyosarcomas in uterine leiomyomas is estimated to be between 0.13 and 0.29%. Little is known of the genetic abnormalities leading to this neoplasia. Loss of p53 function has been implicated in the pathogenesis of many human tumors. We evaluated eight leiomyosarcomas and eight leiomyomas for alterations in exons 5-8 of p53, which are the mutational hotspots for this gene in human malignancies. Genomic DNA of the samples was studied by single-strand conformation polymorphism (SSCP) analysis and positive samples were analyzed by direct sequencing of PCR-amplified products. Each exon was studied individually, and positive controls were used for each exon. Three alterations in a total of eight leiomyosarcoma samples were found; the changes were point mutations (exon 5, codon 165; exon 8, codon 275; exon 8, codon 266). No alterations of p53 could be demonstrated in the eight leiomyoma samples. Our results show for the first time that p53 mutations are frequent in leiomyosarcomas, and one distinguishing difference between benign leiomyomas and some malignant leiomyosarcomas is the acquisition of a p53 mutation.

Number and location of AUUUA motifs: role in regulating transiently expressed RNAs.

Many RNAs coding for either cytokines or oncogenes are unstable and have a short half-life (t1/2). The AUUUA motif is a highly conserved sequence and is repeated three or more times in the 3' untranslated region (3'UTR) of RNAs encoding many of these short-lived cytokines and oncogenes. These sequences can confer instability. In this study, we investigated the role of number and location of AUUUA motifs in stabilization of RNA. We introduced 1xATTTA, 2xATTTA, ATTTTTTTA (second adenosine of 2xATTTA was substituted with a thymidine), 3xATTTA, 5xATTTA, 7xATTTA [AT-rich sequence from granulocyte-macrophage colony-stimulating factor [GM-CSF] gene (AT-62)], and GC-62 (GC sequences were substituted for ATTTA sequences in the 7xATTTA) into the 3'UTR of rabbit beta-globin (R beta G) gene. This construct also contained the neomycin-resistance gene. These expression vectors were transfected into human lung fibroblasts (W138), which constitutively expressed low levels of GM-CSF mRNA. Stable transfectants were selected by growth in G418. Northern blot analysis of actinomycin D-treated, stably transfected cells demonstrated that the number of AUUUA sequences correlated with rapidity of turnover of the chimeric R beta G mRNA. The rank order of stability was GC-62 = 1xATTTA = 2xATTTA (no RNA decay at 4 hours) > 3xATTTA = 5xATTTA (t1/2, 4 hours) > 7xATTTA (t1/2, 2 hours). Stability of mRNA of R beta G also was reduced (t1/2, 2 to 4 hours) when AT-62 was introduced into the second exon of R beta G gene. In these same cells, the t1/2 of GM-CSF RNA was approximately 10 to 15 minutes, suggesting that the AUUUA motifs cannot alone account for the rapid degradation of this cytokine mRNA. Phorbol diesters, including 12-0-tetradecanoyl phorbol 13-acetate (TPA), stabilize a variety of transiently expressed RNAs, including GM-CSF RNA. We found that TPA markedly increased (> 30-fold) the accumulation of GM-CSF RNA. In contrast, TPA was unable to stimulate the levels of the chimeric R beta G when either 1x, 2x, 3x, or 5xATTTA motifs were fused to 3'UTR, or when either AT-62 or GC-62 control sequences were fused to the second exon. The chimeric beta-globin construct with either AT-62 or ATTTTTTTA in the 3'UTR had only an approximately twofold to threefold increase in accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)

The p53 activation domain binds the TATA box-binding polypeptide in Holo-TFIID, and a neighboring p53 domain inhibits transcription.

Antioncogene product p53 is a transcriptional transactivator. To investigate how p53 stimulates transcription, we examined the interaction of p53 with general transcription factors in vitro. We found that p53 binds directly to the human TATA box-binding polypeptide (TBP). We also observed a direct interaction between p53 and purified holo-TFIID, a complex composed of TBP and a group of TBP-associated polypeptides known as TAFs. The p53 binding domain on TBP was mapped to the conserved region of TBP, including residues 220 to 271. The TBP binding domain on p53 was mapped to the p53 activation domain between residues 20 and 57. To analyze the significance of the p53-TBP interaction in p53 transactivation, we compared the ability of Gal4-p53 fusion proteins to bind to TBP in vitro and to activate transcription in transient transfection assays. Fusion proteins which bound to TBP activated transcription, and those that did not bind to TBP did not activate transcription to a detectable level, suggesting that a direct interaction between TBP and p53 is required for p53 transactivation. We also found that inclusion of residues 93 to 160 of p53 in a Gal4-p53 fusion repressed transcriptional activation 100-fold. Consequently, this region of p53 inhibits transcriptional activation by the minimal p53 activation domain. Highest levels of activation were observed with sequences 1 to 92 of p53 fused to Gal4, even though this construct bound to TBP in vitro with an affinity similar to that of other Gal4-p53 fusion proteins. We conclude that TBP binding is necessary for p53 transcriptional activation and that p53 sequences outside the TBP binding domain modulate the level of activation.

Expression of c-jun during macrophage differentiation of HL-60 cells.

Cellular transcription factors are important in the regulation of cellular genes. Recent studies have indicated that a class of cellular genes known as early response genes are important in the control of cellular growth properties. Two of these genes, c-jun and c-fos, play an important role in the control of cellular differentiation. Because the acute myelogenous leukemia cell line, HL-60, is capable of differentiating to either macrophages or granulocytes, it provides a good model to understand differential gene expression. To determine if the modulation of c-jun was important in the differentiation of HL-60 cells to either macrophages or granulocytes, expression of c-jun mRNA was determined by Northern analysis at various times following treatment with a variety of differentiating agents, including 12-tetradeconyl-phorbol 13-acetate (TPA), retinoic acid (RA), dimethyl sulfoxide (DMSO), or 1,25 dihydroxyvitamin D3 [1,25 (OH)2 D3]. Both TPA and 1,25(OH)2D3, which induce HL-60 cells to differentiate to macrophages, resulted in marked increases in c-jun mRNA; while RA and DMSO, which induce HL-60 cells to differentiate to granulocytes, did not greatly alter c-jun mRNA expression. HL-60 cell lines resistant to macrophage differentiation after exposure to either 1,25(OH)2D3 or TPA did not result in increases in c-jun mRNA. These results suggest that elevation of c-jun mRNA in HL-60 cells correlated temporally with differentiation to macrophages. Thus, c-jun may be a critical cellular transcription factor involved in macrophage differentiation.

In vitro action of tumor necrosis factor on myeloid leukemia cells.

We investigated the in vitro action of recombinant tumor necrosis factor alpha (TNF) on the clonal growth of normal and malignant myeloid cells. Clonogenic cells from six of nine myeloid leukemia cell lines were very sensitive to the effects of TNF with 50% of the colonies inhibited (ED50) by concentrations of TNF that ranged between 6 and 150 U/mL. A decrease in DNA, RNA, and protein synthesis and in cloning efficiency occurred within three hours of exposure of HL-60 promyelocytes to TNF. The TNF in combination with recombinant interferons produced an additive or synergistic inhibition of colony formation of HL-60 and THP-1 myelomonoblasts. Normal human CFU-GM are sensitive to TNF (ED50 between 100 and 50,000 U/mL), but their sensitivity to TNF depends on the source of colony stimulating factor (CSF) with T lymphocyte derived GM-CSF (recombinant or natural) partially protecting the CFU-GM from the suppression exerted by TNF (and interferons). In eight of 15 cases the clonogenic myeloid leukemia cells from patients with acute or chronic myeloid leukemia were more sensitive than normal CFU-GM using GM-CSF as a source of colony stimulating activity. Further studies showed that the action of TNF on myeloid leukemia cells probably can be only partially explained by differentiation. Our finding of a possible selective cytotoxicity to leukemic clonogenic cells by TNF suggests that TNF may have value in the therapy of some patients with myeloid leukemia.

A monoclonal antibody cross-reactive with human platelets, megakaryocytes, and common acute lymphocytic leukemia cells.

A cytotoxic monoclonal antibody, CALL1, produced against a human schwannoma tumor was found to react with human platelets, common acute lymphocytic cells (cALL), and lymphoblasts from lymphoid blast crisis of chronic myelocytic leukemia (CML). The hybridoma was repeatedly cloned, and the antibody was considered reactive to a single antigen by absorption tests demonstrating that platelets remove cALL activity and cALL cells absorb platelet activity from the antibody. In addition, chromatofocusing showed that the antibody against platelets and cALL had the same isoelectric point. The CALL1 antibody bound to megakaryocytes but inhibited neither myeloid (CFU-C) nor erythroid (BFU-E) colony formation from bone marrow stem cells. Immunoprecipitation and SDS-gel electrophoresis indicated that CALL1 reacts with a polypeptide of 26,000 daltons.