Lack of mutations in the TP53 tumor suppressor gene exons 5 to 8 in Fanconi's anemia.

The TP53 gene is considered to be a negative regulator of cell growth whose inactivation is an important step in the development or progression of malignancies. Recently, germ line TP53 mutations have been detected in a familial cancer syndrome, the dominantly inherited Li-Fraumeni syndrome. Using single strand conformation polymorphism analysis of PCR products, we looked for TP53 mutations in DNA of patients with Fanconi anemia, an autosomal recessive disease characterized by increased predisposition to neoplasia. We did not find any TP53 mutation in 13 patients, suggesting that this tumor suppressor gene is not directly involved in the cancer susceptibility observed in Fanconi's anemia.

A new Ph1+, bcr cell line derived from a patient with ALL-L1 gained autonomy in culture concomitant to CD23 expression.

A permanent cell line, LEF1, has been established from the cells of an adult suffering from a Philadelphia positive acute lymphoblastic leukemia. The LEF1 cell line was obtained by maintaining peripheral blood cells from the patient in culture on a fibroblast feeder; subsequently, an autonomously growing cell population, independent of that feeder layer, developed. The karyotype of the cell line, 46, t(9;22)(q34;q11), was different from the karyotype at diagnosis which had 53 chromosomes and two Philadelphia chromosomes. Furthermore, compared with the initial leukemic blasts, the immortalized cell had three differences in surface phenotype (CD23+, CD11b-, CD10-). However, molecular studies indicated that the breakpoint in the 3' part of the first intron of the BCR gene was unchanged, confirming the leukemic origin of LEF1. The cell line was shown to be Epstein-Barr virus negative.

Ph1-positive, bcr-negative acute leukemias: clustering of breakpoints on chromosome 22 in the 3' end of the BCR gene first intron.

About 50% of the Philadelphia-positive acute leukemias undergo molecular rearrangements outside the now classical bcr sequence (or M-BCR-1) rearranged in chronic myeloid leukemia (CML). Most of the breakpoints on chromosome 22 have been shown to be clustered in a 10.8-kb region of the first intron of the BCR gene (called bcr2 or m-BCR-1). In this report we examined two cases of Ph1 acute lymphoblastic leukemia in adult patients that exhibited breakpoints in a 5-kb segment of the BCR gene first intron, 16 kb upstream of the previously described cluster, suggesting the possibility of a second minor breakpoint cluster. In addition, the breakpoints on chromosome 9 were located in a region just 5' of the c-abl exon la.

Ph1+bcr- acute leukemias: implication of Alu sequences in a chromosomal translocation occurring in the new cluster region within the BCR gene.

Chromosomal breakpoints on chromosome 22 are located in the first intron of the bcr gene in half of the Philadelphia-positive acute leukemias (Ph1+bcr- AL). We have previously shown that, in these cases, the breakpoints are clustered in the 3' portion of the bcr gene first intron, particularly in a region called bcr2 or m-bcr-1. In order to search for mechanisms underlying the reciprocal chromosome translocation, molecular analysis of breakpoints on chromosome 9 and 22 were performed in a Ph1+bcr- acute lymphoblastic leukemia with bcr2 rearrangement. The comparison of rearranged sequences with their normal counterparts showed that human repetitive Alu sequences were physically linked to the translocation on both chromosomes. In addition an inverted Alu repeat was found 5' to each rearranged Alu sequence on chromosome 22 and 9, with an intervening sequence of 210 and 90bp respectively. This allows to propose a new model, still to be confirmed, of recombination after formation of two hairpin structures which could facilitate the genesis of the chromosomal accident.

Philadelphia-positive acute leukemia: lineage promiscuity and inconsistently rearranged breakpoint cluster region.

Six patients with Philadelphia-positive (Ph1+) acute nonlymphocytic leukemia (ANLL) were studied by morphological, immunological, cytogenetic, and molecular techniques. Seven Ph1+ acute lymphocytic leukemia (ALL) cases were also studied for comparison. Three of ANLL cases were classified in M1, M2, and M4 groups of the FAB nomenclature, while the three other cases do not fit with any FAB subgroup and are described as M0. Immunophenotypical marker studies, double immunolabeling, and combined immunological and cytogenetic studies of metaphases showed that these ANLL expressed several lineage differentiation antigens. Rearrangements of immunoglobulin heavy chain gene (C mu) were detected in the six ANLL cases and in the seven ALL cases studied, as well as, in most cases, rearrangement of T cell receptor beta chain genes and/or T cell rearranging gamma genes. The results favored the assumption that the Ph1 translocation originated from a multipotent stem cell in Ph1+ ANLL. A common t(9;22) translocation was found in all cases, and additional chromosomal abnormalities were present in the six ANLL cases and in five of the seven ALL cases. Molecular studies of bcr gene configuration and c-abl transcription allowed two groups of Ph1+ ANLL to be distinguished. Three cases had bcr rearrangement and c-abl mRNA expression comparable to those reported in Ph1+ chronic myeloid leukemia, while three others had not detectable bcr rearrangement and a 7.2-7.5 kb c-abl mRNA. The existence of Ph1+ ALL with and without classical bcr rearrangement was confirmed.

Epstein-Barr virus-containing epithelial cells from nasopharyngeal carcinoma produce interleukin 1 alpha.

Nasopharyngeal carcinoma (NPC) is a human epithelial cancer that is constantly associated with the Epstein-Barr virus (EBV). Investigations on this tumor have been limited so far by the difficulty of culturing NPC cells for long periods. C15 is an NPC tumor that has been successfully carried in nude mice for greater than 2 yr. C15 cells isolated from the animal were shown to produce a soluble factor with interleukin 1 (IL-1) activity. Its biochemical (Mr, approximately equal to 17,000; pI approximately equal to 5) and immunological properties are identical to those of IL-1 alpha. RNA gel blot analysis showed IL-1 alpha, but not IL-1 beta, transcripts in C15 cells, in sharp contrast to monocytes that express IL-1 beta predominantly. Media from short-term cultures of fresh NPC biopsies also contained a strong IL-1 activity. Several lymphoblastoid cell lines obtained by EBV infection of normal B lymphocytes have been shown to produce IL-1 and use it as an autocrine growth factor. The production of IL-1 by malignant EBV-containing epithelial cells indicates that different types of EBV-infected cells produce IL-1. A relationship might exist between EBV and constitutive production of IL-1. The IL-1 produced by the malignant epithelial cells in vivo could stimulate the development of the pronounced T-cell infiltrate observed in NPC tumors.

c-myc and c-fos expression during interferon-alpha therapy for hairy cell leukemia.

Low-dose interferon-alpha (IFN-alpha) therapy is consistently effective in the treatment of hairy cell leukemia (HCL). In two cases of resistance to IFN-alpha administration, we diagnosed variant HCL, a form of HCL with intermediate features between typical HCL and B cell prolymphocytic leukemia. We tried to distinguish variant and typical hairy cells (HCs) by Northern blot analysis of the oncogenes expressed in vivo. We report that variant HCs contain c-myc transcripts in contrast to typical HCs, whereas c-fos transcripts are detected in both cell types. We also report that the mRNA levels of c-myc are not modified in variant HCs by IFN-alpha treatment, whereas the level of c-fos mRNA is modulated in both types of HCs. Our findings suggest that the failure to modulate c-myc expression in vivo might indicate the limits of low-dose IFN-alpha therapy.

Translation of Schistosoma mansoni antigens in Xenopus oocytes microinjected with mRNA from adult worms.

Oocytes from Xenopus laevis microinjected with RNA isolated from Schistosoma mansoni adult worms translated antigens recognized by sera from infected rats, humans, and from immunized rabbits. The pattern of immunoprecipitated proteins analysed by SDS-polyacrylamide gel electrophoresis was species specific in rats. Serum from infected Fischer rats recognized antigens of 20, 27 and several bands in the 50-60 kDa range whereas serum from infected Brown Norway rats also immunoprecipitated major bands at 29, 43 and 100 kDa. Human infection sera gave a very variable pattern of immunoprecipitation not apparently dependent on the patients' age. At least 20 different antigenic species could be identified ranging from 14 to 150 kDa. Some S. mansoni antigenic proteins could be isolated from the membrane fraction of the oocytes whereas notably the 29 kDa band was present mainly in the soluble fraction. N-Glycosylation of S. mansoni antigens occurred as evidenced by the effects of tunicamycin treatment and concanavalin A binding. A multiple series of bands between 50 and 60 kDa, present in the membrane fraction, were glycosylated and secreted from the oocytes. Monoclonal antibodies to larval stage surface antigens failed to immunoprecipitate oocyte translation products, but sera absorbed with live schistosomula identified at least three putative surface antigens of 100, 43 and 29 kDa. However, the 29 kDa molecule was neither synthesized into membranes, nor secreted from oocytes.

Messenger RNA extracted from Schistosoma mansoni larval forms codes for parasite antigens when translated in vitro.

Intact messenger RNA was extracted from three stages of development of the parasite Schistosoma mansoni, cercariae, 3 h (mechanically prepared) schistosomula and adults. Some of the in vitro translation products are precipitable with immune rat and human serum showing that these sera recognize protein determinants. Whereas the patterns of the total translation products show major proteins expressed at all three developmental stages, immunoprecipitation delineates patterns unique to each stage. Serum from chronically infected patients is likely to preferentially recognize proteins expressed by the adult parasite yet precipitates more products of translation of cercarial RNA than of adult RNA, suggesting that many of the same epitopes are already coded by cercarial messenger RNA. It seems, however, that antigens appear on lower molecular weight species in the adult. The experiments described here open the way to cloning the genes of antigens from S. mansoni.

Murine leukemia virus-associated cell surface antigens in rats neonatally infected with Gross murine leukemia virus.

The inoculation of newborn W/F, Lew, AS and DA rats with Gross murine leukemia virus (G-MuLV) resulted in the prompt appearance of cells with viral protein antigens (VPA) on their surfaces. These were first found in the bone marrow and spleen and later in the thymus gland. As the animals developed, the VPA-positive population expanded and the intensity of the fluorescence increased. In the spleen, the cells with the strongest fluorescence had the properties of T-cells, but in both spleen and bone marrow low levels of VPA were found on non-T-cells. The VPA-positive population expanded long before malignant cells could be detected and, in most animals, the entire T-cell compartment became antigen-positive. These animals were unable to respond to G-MuLV antigens and many eventually developed leukemia. However, some animals apparently broke the tolerance that followed neonatal infection and eliminated VPA-positive cells from their tissues