Sequence of 10q24 locus surrounding the HOX11 oncogene reveals a new gene HUG1 expressed in a T-ALL cell line.

HOX11 is a gene encoding a homeobox protein which is found to be deregulated in T-cell acute lymphoblastic leukaemia (T-ALL). As a basis for studying the mechanism of deregulation of HOX11 expression in leukaemia, the locus containing the HOX11 proto-oncogene at 10q24 was cloned from a genomic P1 Artificial Chromosome (PAC) library. The PAC clone with an insert size of 120kb was isolated and mapped by restriction analysis. A series of contiguous subclones were then obtained which span 20kb surrounding the HOX11 gene. These subclones were used to sequence across the entire 20kb region to the 3' boundary of the PAC insert. This work provides for the first time the full intron and 5' non-coding sequences of the HOX11 gene which will aid the identification of novel transcriptional control elements which may be involved in silencing HOX11 expression in normal cells. The sequence information was also used to search for novel large open reading frames (ORFs). One such ORF (1.1kb) would encode a protein of at least 39kDa. This basic protein (pI, 12.5) would be very proline rich and could potentially encode a novel transcription factor. In order to establish if this ORF corresponds to a bona fide transcribed gene, RT-PCR analysis was performed. The mRNA for this protein is expressed in the T-ALL cell line Jurkat and has been designated HUG1, for HOX11 Upstream Gene.

A new pineoblastoma cell line, PER-480, with der(10)t(10;17), der(16)t(1;16), and enhanced MYC expression in the absence of gene amplification.

Pineoblastoma is a rare, but highly malignant tumor of the central nervous system (CNS) in children and is classified as a central primitive neuroectodermal tumor (PNET). Despite notable recent advances in understanding the molecular genetic basis of malignancies, the pathogenesis of PNETs remains enigmatic. There is scant information on the cytogenetics of PNETs arising in the pineal gland and the only three reported cases did not show any common aberrations. Here we report the establishment and characterization of a new pineoblastoma cell line, PER-480. The biopsy material and the cell line were characterized using light and electron microscopy and immunohistochemical analyses. The cell line was examined for expression of cell surface markers using a panel of monoclonal antibodies and by cytogenetic analysis. MYC family genes were studied at the DNA, RNA, and protein level. Cell line PER-480 showed neuronal differentiation and the karyotype demonstrated two abnormalities, a der(10)t(10;17) and a der(16)t(1;16). An intriguing finding is that all three pineoblastoma cell lines established in our laboratory, PER-452, PER-453, and PER-480, showed enhanced expression but not amplification of a member of the MYC family of proto-oncogenes. Cell line PER-480 reported here will be useful for the further investigation of the molecular genetic basis of central PNETs.

Deletions of the p16 gene in pediatric leukemia and corresponding cell lines.

The p16 gene (MTS1 or CDK4I) encoding an inhibitor of cyclin-dependent kinase 4 (cdk4), has been reported to be deleted in various tumor cell lines, including lines derived from leukemic cells. The reported frequency of p16 gene loss is much higher in established cell lines than in primary tumor specimens. We investigated the status of the p16 gene in pediatric leukemias using 12 established cell lines of differing phenotypes and their corresponding primary leukemic cells. Six of 12 cell lines, including acute lymphoblastic leukemia (ALL) lines of T-cell (three of four), of precursor-B cell (two of four) and of mixed phenotype (one of four), showed homozygous deletion of the p16 gene using PCR and Southern blotting. Comparison of the cell lines with their corresponding primary leukemic cells clearly showed that in all 12 paired samples there were identical findings with respect to the presence or absence of the p16 gene, demonstrating that loss of the gene was a feature of the primary leukemic cells. This is the first study to show this correlation using a panel of paired samples, indicating that p16 gene deletions were not an artifact of in vitro cell culture. Furthermore, the survival of ALL patients with p16 gene deletions was significantly inferior to those without deletions, suggesting that this genetic alteration may be a clinical prognostic factor.

HOX11 expression in pediatric acute lymphoblastic leukemia is associated with T-cell phenotype.

Based on cytogenetic analysis, chromosomal translocations involving band 10q24 occur in 4-7% of T-cell acute lymphoblastic leukemia (T-ALL). The HOX11 gene is located in this chromosomal band and is activated by translocations t(10;14) (q24;q11) and t(7;10) (q35;q24). Ectopic expression of the HOX11 gene appears to be involved in the development of T-cell tumors. The aim of this study was to determine the frequency of HOX11 activation in pediatric ALL patients and to correlate gene expression with ALL immunophenotype. None of 53 B-lineage ALLs was positive for HOX11 expression, however, Northern blot and RT-PCR analysis revealed that four of 12 T-ALLs (33%) showed expression of the gene. In order to assess whether HOX11 expression is present in other pediatric malignancies we examined a panel of 20 tumor cell lines established from solid tumors and leukemias, but none of them showed expression of HOX11. Using our RT-PCR method we confirmed that HOX11 expression is not detectable in normal T-cells. These findings indicate that HOX11 expression in pediatric ALL is exclusive to T-ALL and does not occur in B-lineage ALL. The frequency detected by molecular techniques was significantly higher than the frequency reported in the literature based on cytogenetic analysis. These results support the notion that ectopic expression of the HOX11 homeobox gene is a crucial step in T-cell tumorigenesis.

New translocation t(2;13)(p12;q34) and rearrangement of the MLL gene in a childhood leukemia cell line.

Here we report the case of a 7-month-old boy who presented with biphenotypic acute leukemia, but with leukemia cells of B-cell phenotype present at the time of relapse. Two cell lines were derived from bone marrow specimens obtained at relapse, and immunophenotyping and analysis of antigen receptor gene configuration revealed concordance between the patient's leukemic cells and the cell lines. Cell line PER-377 shows a new chromosomal abnormality, t(2;13)(p12;q34), a molecular rearrangement at chromosome band 11q23 in the absence of a cytogenetically detectable abnormality of this band, and deletion of the genes for IGK.

Enhanced MYCN expression and isochromosome 17q in pineoblastoma cell lines.

We have established two cell lines, PER-452 and PER-453, from an 8-month-old girl with an extensive pineoblastoma. Characterization of these lines revealed that the proto-oncogenes MYC and MYCN were not amplified, but both cell lines showed MYCN expression comparable to a cell line with 200-fold MYCN amplification. Both cell lines contained an i(17q). These results support the concept that pineoblastomas belong to a larger group of primitive neuroectodermal tumors of the central nervous system. These two cell lines provide a unique opportunity to investigate the molecular genetic mechanisms underlying these neoplasms further.

A new human cell line for the molecular dissection of the requirement for interleukin-1 in the production of interleukin-2 by immature T cells.

An immature human T cell line, PER-117, can be induced to secrete interleukin-2 (IL-2). In contrast to mature T cells or the Jurkat cell line, PER-117 cells require interleukin-1 (IL-1) for optimal IL-2 secretion, in addition to calcium ionophore and phorbol 12-myristate 12-acetate (PMA). These requirements mirror the conditions reported to be optimal for normal immature T cell receptor (TCR) negative thymocytes. IL-1 did not substitute for either of the other signals required for IL-2 production, i.e., calcium ionophore or PMA, suggesting that IL-1 activates pathways different from those elicited by the two other stimuli. For optimal effect, IL-1 needed to be provided at the same time as the two other signals. Significantly, a signal provided by a low concentration of PMA (not leading to IL-2 induction by itself in the presence of calcium ionophore) was necessary. The studies reported here provide the first evidence that three signals are required for optimal IL-2 production in a human immature T cell line. PER-117 cells produce substantial levels of IL-2 and thus provide a model to study stage-specific signal transduction and transcriptional activation of the IL-2 gene in IL-1 responsive immature thymocytes.

Requirements for the induction of the interleukin-2 receptor complex in a human pre-T-cell line.

Cell line PER-117 is a T-cell receptor negative human T-cell line that can be induced to express a functional interleukin-2 receptor (IL-2R). Recombinant interleukin-1 (IL-1) as well as certain combinations of inducer substances could be shown to stimulate the expression of the p55 (alpha)-chain of the IL-2R in PER-117 cells. The synergistic increases in IL-2R alpha expression were demonstrated at the cell surface as well as at the mRNA level. The results suggested that in PER-117 cells IL-1 appears to induce expression of the alpha-chain by pathways that are different to activation via protein kinase C (PKC), and that drug-induced cyclic AMP (cAMP) activation did not substitute for IL-1. We found that the regulation of mRNA for IL-2R beta (p75) differed significantly from that seen for IL-2R alpha. Moreover, the requirements for IL-2R alpha induction determined for this cell line differ from other human cell lines, which may reflect that there are distinct requirements for activation depending on the stage of differentiation and/or lineage of the cells. The PER-117 cell line provides a unique model to examine further the mechanism leading to induction of a functional IL-2R at an early stage of human T-cell differentiation.

Functional interleukin-2 receptor on a Tac negative human leukaemia T-cell line.

Cell line PER-423 was derived from the cells of a patient with an immature acute T-lymphoblastic leukaemia and the growth of this human cell line is strictly dependent on interleukin-2 (IL-2). PER-423 cells express the p75 (beta) subunit of the IL-2 receptor (IL-2R beta), while the p55 chain (IL-2R alpha) is not detectable by immunofluorescence. The analysis of the IL-2R revealed that it is of intermediate affinity and the median effective IL-2 concentration for PER-423 cells (EC50 value) was determined to be 1.44 +/- 0.29 nM. Chemical crosslinking studies showed that the receptor consists of one polypeptide of approximately 95 kDa as well as a doublet of 70 kDa and 60 kDa and does not include the IL-2R alpha-chain. The steady-state mRNA level for the p75 subunit was similar to that present in a cell line expressing an IL-2R alpha+ beta+, while only traces for the alpha-chain were detectable. PER-423 cells can be induced to express the alpha-chain of the IL-2R on the cell surface, concomitant with a much reduced EC50 level. Since cell line PER-423 is functionally dependent on IL-2, it provides an ideal model for IL-2 signal transduction studies and for investigations focusing on the requirements for ligand binding vs activation.

Three neuroblastoma cell lines established from consecutive samples of one patient which show distinct morphologic features, MYCN amplification, and surface marker expression.

Three neuroblastoma cell lines established from tumor samples obtained from one patient are described. The three lines were derived from bone marrow aspirates taken at diagnosis, and 13 and 15 months later. The origin of the cell lines PER-106, PER-107, and PER-108 from malignant neuroblasts was confirmed by electron microscopy studies, surface marker analysis, and assessment of MYCN amplification. Cell lines PER-107 and PER-108, which were established from tumor samples obtained at the time of progressive disease, have significantly shorter doubling times than PER-106 and grow mainly substrate-adherent, while cell line PER-106 (established from sample obtained at diagnosis) consists of small round neuroblastic cells which form large aggregates in suspension culture. The electron microscopy studies revealed distinctive neuroblast-like ultrastructure in all cell lines. The MYCN copy number was amplified (greater than 10 copies) in the established cell lines and in the fresh tumor samples and the relative abundance of MYCN RNA in the cell lines correlated roughly with the extent of the MYCN gene amplification. However, distinct phenotypic differences can be demonstrated among these three lines, which provide a model for the further examination of this highly malignant tumor. Detection of MYCN amplification by chromosomal in situ hybridization was performed on this set of cell lines, as reported by McRobert et al. (this issue, pages 128-134).

Characterization of an interleukin-2 dependent human leukemic cell line, PER-315, with an immature T cell phenotype which does not express the tac antigen.

Cell line PER-315 was established from a bone marrow sample of a 5-year-old boy diagnosed with acute lymphoblastic leukemia (ALL) of T cell lineage. PER-315 cells express the surface markers present on immature thymocytes, express cytoplasmic CD3, and their growth is dependent on interleukin-2 (IL-2). Hence, this cell line represents a new type of precursor T-ALL, which is IL-2 dependent. Assessment of the T cell receptor rearrangements confirmed the clonal origin of cell line PER-315, and comparison with the patient's leukemia cells revealed an identical pattern. PER-315 cells show strong cytotoxicity against cell lines K562, Daudi, and Molt-4. They do not express the Tac antigen, but bind IL-2 with a Kd of 650 pM. Since PER-315 cells represent immature thymocytes, this new cell line may provide a model to further investigate the IL-2 receptor structure present at this stage of T cell differentiation.

HLA-DR and -DQ DNA genotyping in insulin-dependent diabetes patients in South India.

Restriction fragment length polymorphisms (RFLPs) of the HLA-DR beta, -DQ alpha, -DQ beta, and -DX alpha genes have been examined in South Indian diabetic patients and controls. The DR. DQ linkage arrangements in South Indians were shown to be different for DR2, DR4, and DRw6 from those commonly seen in Europeans, so that localization of the primary disease-promoting gene in IDDM could be attempted. This study clearly implicates at least one DQ beta allele in the pathogenesis of IDDM.

HLA, complement C2, C4, properdin factor B and glyoxalase types in South Indian diabetics.

A series of diabetic patients from 3 centres in South India have been tested for HLA A, HLA B, BF, C2, C4A, C4B and GLO types. For insulin-dependent diabetes mellitus (IDDM) patients there was a significant increase in HLA B8, of BF F and decrease of C4 A6. No significant variation in HLA, BF, C2 or GLO frequencies was found in non-insulin-dependent diabetes mellitus (NIDDM) patients, but there was a significant decrease in C4B 1 and an increase in C4B 2. The HLA and BF association in South Indian IDDM patients is very different from that reported previously in North India.

Population genetics of the vitamin D binding protein (GC) subtypes in the Asian-Pacific area: description of new alleles at the GC locus.

Isoelectric focussing (IEF) in thin layer polyacrylamide gels pH range 4-6.5 has been used to analyse the GC phenotypes of 4233 individuals from 28 different population groups in the Asian, Pacific, and Australian area. Because this technique reveals subtypes of the common GC*1 allele, there is almost a two-fold increase in the mean heterozygosity at the GC locus using IEF compared with conventional electrophoresis. The highest frequency (above 50%) of the GC*1S allele was encountered in Indian populations, reflecting genetic affinities with Europeans. By comparison, east and south east Asians are unique offing maximum values of the GC*1F allele (50%). With the exception of a few Pacific populations which show similar frequencies to east Asians, all other groups in the Pacific area, including Australia, have values of GC*1F similar to GC*1S ranging from 27% to 40%. The GC*2 frequency in most populations varies from 20% to 30%. However, some Polynesian groups have values up to 40% and Australian Aborigines less than 10%. Among other alleles, GC*1A1 is found to be widely distributed among Australian Aborigines and Melanesians and occurs sporadically in Polynesians, Micronesians, and in the Lesser Sunda Islands. Four new alleles, GC*1C24, GC*1C35 Aborigine, GC*1A21, and GC*1A22 are described. The gene frequency data at the GC locus has been used to calculate Nei genetic distances between the populations studied.

Distribution of complement factors Bf, C2 and C6 in the Western Pacific.

Complement components Bf, C2 and C6 have been typed in various Micronesian, Polynesian, Melanesian and Indian populations of the Western Pacific. BfS and BfF gene frequencies are heterogeneous throughout the region but the number of alleles in the Bf system is restricted. C2 is also restricted with the C22 gene frequency from 0-1.3% in all groups except Fijian Indians where it is 4.8%. The C6A and C6B frequencies of Polynesians, Melanesians and Fijian Indians are within the narrow range of frequencies reported for these alleles from other parts of the world. In the Micronesians of Nauru, a third polymorphic allele (C6Nauru) occurs together with a number of rare alleles (C6R). The Nauruan C6 gene frequencies are C6A 44.6%, C6B 45.2%, C6Nauru 6.7% and C6R 3.5% C6Nauri is present in low frequencies in most other Western Pacific populations and C6R variants occur in Polynesians.

Distribution of complement C'2 and C'6 types in Australian cases of diabetes mellitus.

A series of patients with juvenile onset diabetes (IDDM) and mature onset diabetes (NIDDM) have been typed for genetic variants of two sets of complement factors. For the HLA-linked C'2 system, there was found a significant increase of the C'2 2-1 type in IDDM compared with NIDDM patients or healthy controls. No such increase in any phenotype was observed for the non-HLA-linked C'6 system. These observations emphasize again the genetic distinction between IDDM and NIDDM, and the role of chromosome 6 in controlling susceptibility to the insulin-dependent form of the disease.