Cloning and characterization of the SIL promoter

The SIL gene undergoes a site-specific rearrangement with the SCL gene in 25% of patients with T cell acute lymphoblastic leukemia (ALL). The functional result of this rearrangement is that the SIL regulatory elements aberrantly drive expression of the SCL gene. We have cloned and sequenced the human SIL promoter, cloned a murine homolog, found the sequence to be highly conserved, and defined a minimal promoter region. Both the cloned murine and human sequences were found to be highly active in either human or murine cells. SCL mRNA, driven by a cloned SIL promoter, could be downregulated by DMSO in stably transfected F4-6 murine erythroleukemia cells. The SIL promoter was found to be partially unmethylated in proliferating tissues, in which it is highly expressed, and more highly methylated in post-mitotic tissues, in which SIL is not expressed. The isolation of the SIL promoter provides an important tool for the study of both the SIL gene expression as well as the role of the SIL promoter in leukemogenesis.     

Molecular cloning of two CD7 (T-cell leukemia antigen) cDNAs by a COS cell expression system.

The human CD7 antigen (gp40) is a cell surface glycoprotein found on thymocytes and mature T-cells. It is one of the earliest antigens to appear on cells of the T-lymphocyte lineage, and the most reliable clinical marker of T-cell acute lymphocytic leukemia. This report describes the isolation and nucleotide sequence of a full length CD7 cDNA, and of a cDNA for an unusual intron-bearing precursor. The DNA sequence of the clone predicts a highly glycosylated membrane protein with homology to members of the immunoglobulin superfamily, and no relationship to known oncogenes. Over-expression of CD7 RNA was observed in only one T-cell tumor line, and genomic DNA rearrangement was not observed in any lines. Prompted by a recent suggestion that CD7 plays a role in IgM binding, COS cells expressing CD7 were tested and found not to bind IgM or IgM immune complexes.     

Tissue specific expression and chromosomal mapping of a human UDP-N-acetylglucosamine: alpha1,3-d-mannoside beta1, 4-N-acetylglucosaminyltransferase

A human cDNA for UDP- N -acetylglucosamine:alpha1,3-d-mannoside beta1,4- N- acetylglucosaminyltransferase (GnT-IV) was isolated from a liver cDNA library using a probe based on a partial cDNA sequence of the bovine GnT-IV. The cDNA encoded a complete sequence of a type II membrane protein of 535 amino acids which is 96% identical to the bovine GnT-IV. Transient expression of the human cDNA in COS7 cells increased total cellular GnT-IV activity 25-fold, demonstrating that this cDNA encodes a functional human GnT-IV. Northern blot analysis of normal tissues indicated that at least five different sizes of mRNA (9.7, 7.6, 5.1, 3.8, and 2.4 kb) forGnT-IV are expressed in vivo. Furthermore, these mRNAs are expressed at different levels between tissues. Large amounts of mRNA were detected in tissues harboring T lineage cells. Also, the promyelocytic leukemia cell line HL-60 and the lymphoblastic leukemia cell line MOLT-4 revealed abundant mRNA. Lastly, the gene was mapped at the locus on human chromosome 2, band q12 by fluorescent in situ hybridization.     

Induction of anti B-cell malignance CTL response by subfamily-shared peptides derived from variable domain of immunoglobulin heavy chain

The variable domain of immunoglobulin heavy chain (Ig HV) is well-characterized tumor associated antigen expressed in B-cell malignancies, which may function as a T-cell target. However, T-cell epitopes derived from shared framework regions (FRs) of each IgHV subfamily capable of inducing cytotoxic T lymphocytes (CTLs) against the B-cell malignancy, have not been identified. Using the specific PCR primers of seven IgHV gene subfamilies, we amplified the IgHV gene rearrangement for 108 cases of B-cell acute lymphoblastic leukemia (B-ALL) patients. The IgHV gene rearrangement fragments of B-ALL patients were directly sequenced then classified into seven different subfamilies. The T-cell epitopes encoded by the IgHV gene in the B-ALL patients were predicted by SYFPEITHI and BIMAS programs and compared with those from 56 representative germline IgHV sequences in the genebank. For the HLA-A*0201 locus, we found 1 or 2 top score shared epitopes from each subfamily and got 12 epitopes altogether. Results showed that ten of them were in the FRs. Using an antigen-specific T-cell expansion system, we generated the peptide-special CTLs in vitro, which were capable of killing B lymphoma cell lines that belonged to the same IgHV subfamily in a peptide-specific and HLA-restricted manner. Furthermore, we proved that the cytotoxicity of CTLs was IgHV subfamily-specific. These data indicate possible immunotherapy approaches for B-cell malignances patients based on IgHV gene subfamilies.     

Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.     

Apoptosis Induction in Human Lymphoma and Leukemia Cell Lines by Transfection via Dendrosomes Carrying Wild-Type p53 cDNA

The efficiency of dendrosomes (novel dendritic spheroidal nanoparticle gene porters) were assessed in transferring wild-type p53 cDNA into two human acute lymphoblastic leukemia cell lines (MOLT-4 and CCRF-CEM) derived from T cells and erythroleukemic cell line K562. Flow cytometric studies showed a 65% and 45% enhancement in apoptosis and necrosis of K562 and CCRF-CEM cells transfected with complex of dendrosomes and wild-type p53 cDNA in comparison to controls. The cytotoxicity studies on T lymphoma cells revealed that dendrosomes have a low cytotoxicity in comparison to lipofectin.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line kappa light chain genes and germ-line T-cell receptor beta- and gamma-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Spontaneous cytotoxicity of human lymphoblast cell lines mediated by normal peripheral blood lymphocytes. I. Differential susceptibility of T-versus B-cell lines.

Human lymphoblast cell lines of B- and T-cell origin have been tested for their ability to serve as targets in a 4-hr ⁵¹Cr release microcytotoxicity assay using normal human peripheral blood lymphocytes as effector cells. Cell lines of T-cell origin were susceptible to lysis in this assay by effector lymphocytes from all normal donors tested. Cell lines of B-cell origin were repeatably lysed by normal lymphocytes from some, but not all donors. Spontaneous cytotoxicity of B-cell lines, when observed, was also quantitatively less than was obtained using T-cell lines as targets. One cell line (RPMI-7666), of B-cell origin, was not susceptible to spontaneous cytotoxicity by almost all of the normal lymphocyte effectors tested. Lymphocytes from patients with acute lymphoblastic leukemia in remission were less capable of effecting lysis in this assay.     

Breakpoint junctions of chromosome 9p deletions in two human glioma cell lines.

Interstitial deletions of the short arm of chromosome 9 are associated with glioma, acute lymphoblastic leukemia, melanoma, mesothelioma, lung cancer, and bladder cancer. The distal breakpoints of the deletions (in relation to the centromere) in 14 glioma and leukemia cell lines have been mapped within the 400 kb IFN gene cluster located at band 9p21. To obtain information about the mechanism of these deletions, we have isolated and analyzed the nucleotide sequences at the breakpoint junctions in two glioma-derived cell lines. The A1235 cell line has a complex rearrangement of chromosome 9, including a deletion and an inversion that results in two breakpoint junctions. Both breakpoints of the distal inversion junction occurred within AT-rich regions. In the A172 cell line, a tandem heptamer repeat was found on either side of the deletion breakpoint junction. The distal breakpoint occurred 5' of IFNA2; the 256 bp sequenced from the proximal side of the breakpoint revealed 95% homology to long interspersed nuclear elements. One- and two-base-pair overlaps were observed at these junctions. The possible role of sequence overlaps, and repetitive sequences, in the rearrangement is discussed.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Gene expression and prostate specificity of human prostatic acid phosphatase (PAP): evaluation by RNA blot analyses.

A fragment of a complementary DNA (cDNA) clone for human prostatic acid phosphatase (PAP) (EC 3.1.3.2.) was used to study the expression of corresponding mRNA in human tissues. The specificity of its expression in benign prostatic hyperplasia (BPH) and prostatic carcinoma tissues were indicated in RNA blot analyses. The PAPcDNA probe did not recognize any specific mRNAs in RNAs extracted from human liver cancer, lung cancer, pancreatic cancer, placenta, breast cancer cells (MCF-7), mononuclear blood cells or acute promyelocytic leukemia cells (HL-60), according to Northern blot analysis. mRNA for PAP was detected in the androgen-dependent human prostatic cancer cell line LNCaP, but not in the androgen-insensitive human prostatic cancer cell line PC-3. In contrast, lysosomal acid phosphatase (LAP) mRNA was detected in both of these human prostatic cancer cell lines. Our findings indicate a high specificity for the PAP gene in prostatic tissue. The mean abundance for the PAPmRNA expression was 0.26 for prostatic carcinoma samples (n = 11) and 0.46 for BPH samples (n = 8) according to slot-blot analysis. The differences observed between the different categories of prostatic tissue in PAPmRNA abundances call for additional studies on regulation of its expression.