The human PD-1 gene: complete cDNA, genomic organization, and developmentally regulated expression in B cell progenitors.

We report the complete cDNA sequence and the genomic structure of the human PD-1 homologue. An analysis of the expression pattern of the human PD-1 gene (hPD-1) and the murine PD-1 gene (mPD-1) in developing bone marrow B-lineage cells was also undertaken. The full length hPD-1 cDNA is 2106 nucleotides long and encodes a predicted protein of 288 amino acid residues. The hPD-1 and mPD-1 genes share 70% homology at the nucleotide level and 60% homology at the amino acid level. Four potential sites for N-linked glycosylation are conserved, as are a stretch of amino acids between two cysteine residues resembling a V-set immunoglobulin domain, and another region containing a motif similar to an immunoreceptor tyrosine-based inhibitory motif. Isolation of the genomic locus of the hPD-1 gene reveals that the gene is composed of five exons located on human chromosome 2 at band q37. The 5' flanking region lacks TATA and CAAT cis-acting elements, but includes a number of potential transcription factor binding sites and a dominant transcription start site. The mPD-1 gene was preferentially expressed in pro-B cells from murine adult bone marrow. Although hPD-1 was not preferentially expressed in pro-B cells from human fetal bone marrow, treatment of isolated pro-B cells with interleukin-7 resulted in a dramatic increase in expression. These data suggest that PD-1 may play a role in B-cell differentiation during the pro-B cell stage.

Activation-induced expression of human programmed death-1 gene in T-lymphocytes.

The Programmed Death-1 (PD-1) gene is a member of the immunoglobulin superfamily of genes. Murine PD-1 mRNA expression has been shown to correlate with activation-induced apoptosis in a mouse T-cell hybridoma cell line and in murine thymocytes. Here we report that expression of the human homolog, hPD-1, seems to correlate with activation of T lymphocytes rather than apoptosis. We observed a time-dependent upregulation of hPD-1 mRNA and protein levels in Jurkat cells during phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA)-induced differentiation. Human PD-1 protein was also induced during lectin-stimulated activation of human peripheral blood mononuclear cells. Additionally, TPA stimulation of Jurkat cells induces tyrosine phosphorylation of hPD-1, putatively on its cytoplasmic tail signal transduction motif. These data suggest a role for hPD-1 during activation and differentiation of T-lymphocytes.

Identification of an altered immunoglobulin heavy-chain gene rearrangement in the central nervous system in B-precursor acute lymphoblastic leukemia.

In B-precursor acute lymphoblastic leukemia (ALL), the nucleotide sequence of the complementarity determining region III (CDRIII) in the rearranged immunoglobulin heavy chain gene (IgH) has been used as a molecular fingerprint to identify the leukemic cells. In a child with B-precursor ALL without central nervous system (CNS) disease at diagnosis and a subsequent isolated CNS relapse, we examined the stability of the rearranged IgH by comparing the nucleotide sequences of the CDRIII in the leukemic cells from the marrow at diagnosis to the sequences in the leukemic cells from the cerebrospinal fluid at relapse. Whereas two of the three IgH sequences isolated from the leukemic cells at CNS relapse were identical to sequences originally isolated from the marrow lymphoblasts at diagnosis, the third CNS sequence was similar but not identical to the third marrow sequence. The third IgH sequence identified in the CNS differed from the marrow sequence only at the variable gene segment adjoining the CDRIII. Using a detection method based on the polymerase chain reaction, the altered IgH sequence identified in the leukemic cells from the cerebrospinal fluid was noted to be present in the CNS at a higher frequency than the related diagnostic sequence and was not detected in the marrow either at diagnosis or at CNS relapse. These findings indicate that the clonal pattern of leukemia in the CNS may differ from that in the marrow.

VH gene rearrangement events can modify the immunoglobulin heavy chain during progression of B-lineage acute lymphoblastic leukemia.

The presence of multiple VHDJH joinings in upwards of 30% of acute lymphoblastic leukemias (ALL) suggests a relative instability of the rearranged immunoglobulin heavy chain (IgH) gene, but the mechanisms involved are not completely understood. An investigation of the structure of the VHDJH joinings using complementarity determining region (CDR)3 polymerase chain reaction (PCR) in 12 leukemias at both diagnosis and relapse indicates that this instability may increase as a function of time. In only one of seven cases in which relapse occurred within 3 years from diagnosis was a new VHDJH joining identified and this coexisted with the original diagnostic joining. Most strikingly, new VHDJH joinings were identified in four of five cases in which relapse occurred more than 5 years from diagnosis. In this latter population, the instability of the joinings was generated from VH----VH gene replacement events in two cases, since the new joinings retained the original DJH sequences and partial N region homology at the VHD junction, and probably in a third case from a VH gene rearrangement to a common DJH precursor. Furthermore, in five of 23 (21.7%) additional cases studied at diagnosis, subclones were identified that had similar modifications of the VH-N region. These data indicate that VH gene replacement events and VH gene rearrangements to a common DJH joining contribute to the instability of the VHDJH joining in ALL. This phenomenon should be taken into consideration in those methodologies that exploit IgH rearrangements for detection of minimal residual disease.

Short course high dose mitoxantrone with high dose cytarabine is effective therapy for adult lymphoblastic leukemia.

Mitoxantrone is effective in the treatment of acute myelogenous leukemia and, in combination with either vincristine-prednisone or cytarabine (HiDAc), it is also effective in acute lymphoblastic leukemia (ALL). As mitoxantrone exhibits a steep dose-response curve against ovarian cancer cells and acute myeloid leukemia cells in vitro, we evaluated the safety and efficacy of high dose mitoxantrone with HiDAc in the treatment of ALL. Patients received mitoxantrone 20-37.5 mg/m2 daily for 2 days or 1 dose of 40-80 mg/m2 and HiDAc 3 g/m2 over 3 h once daily for five doses. All eight of the patients with previously untreated disease and eight of ten patients with ALL in relapse achieved complete remission (CR). The untreated patients included two with Philadelphia positive ALL who also achieved CR (one after one course of mitoxantrone-HiDAc, and one after one course of mitoxantrone-HiDAc followed by one additional dose of vincristine and daily prednisone). Seven of the eight previously untreated patients who achieved CR are still in remission. The one T-cell ALL has relapsed at 2 months after CR. The toxicity was acceptable. The regimen thus induces a remission rate equivalent to that of traditional vincristine-prednisone. The 'quality of remission' may be superior, and this therapy should be explored as a primary induction therapy in patients with ALL.

Detection of minimal residual disease in leukemic patients with the t(10;14)(q24;q11) chromosomal translocation.

Early relapse and minimal residual disease during clinical remission was examined in two patients having acute T-cell leukemia/lymphoma with the t(10;14)(q24;q11) chromosomal translocation. Molecular probes which can detect T-cell receptor alpha/delta clonal rearrangements and a TCL-3 probe which can detect the clonal rearrangement due to the chromosomal translocation failed to detect the leukemic clones during clinical remission by Southern filter hybridization. However, application of the polymerase chain reaction technology in amplification of the t(10;14)(q24;q11) chromosomal juncture during clinical remission permitted us to increase the detection level of neoplastic cells up to 1 leukemic cell/125,000 normal cells using 1 microgram of DNA. Amplified junction fragments were detected in both patients. In one case, during the period of clinical remission no amplified fragments were detected.

Involvement of the TCL5 gene on human chromosome 1 in T-cell leukemia and melanoma.

We analyzed a t(1;14)(p32;q11) chromosomal translocation in a human lymphohemopoietic stem cell line derived from a patient with acute T-lymphoblastic leukemia. The chromosomal joining on the 1p+ chromosome occurred at the T-cell receptor delta diversity (D delta 2) segment, and the reciprocal chromosomal joining on the 14q- chromosome occurred at the T-cell delta diversity segment D delta 1. The involvement of delta diversity segments at the translocation junctions suggests that the translocation occurred during an attempt at D delta 1-D delta 2 joining in a stem cell. The segment of chromosome 1 at band p32, adjacent to the chromosomal breakpoint, encodes a transcriptional unit designated TCL5 (T-cell leukemia/lymphoma 5). The differential expression of the TCL5 RNA transcripts in this lymphohemopoietic stem cell line relative to several other T- and B-cell lines suggests that TCL5 gene expression is an integral event in the pathogenesis of the T-cell leukemia. Rearrangement of the TCL5 locus in a human melanoma cell line carrying a del(1p32) further implies that the TCL5 gene may play a role in malignant transformation.

Clustering of breakpoints on chromosome 10 in acute T-cell leukemias with the t(10;14) chromosome translocation.

The T-cell receptor (TCR) alpha/delta chain locus on chromosome 14q11 is nonrandomly involved in translocations and inversions in human T-cell neoplasms. We have analyzed three acute T-lymphoblastic leukemia samples carrying a t(10;14)(q24;q11) chromosome translocation by means of somatic cell hybrids and molecular cloning. In all cases studied the translocation splits the TCR delta chain locus. Somatic cell hybrids containing the human 10q+ chromosome resulting from the translocation retain the human terminal deoxynucleotidyltransferase gene mapped at 10q23-q24 and the diversity and joining, D delta 2-J delta 1, regions of the TCR delta chain, but not the V alpha region (variable region of the TCR alpha chain), demonstrating that the split occurred within the V alpha-D delta 2 region. Molecular cloning of the breakpoint junctions revealed that the TCR delta chain sequences involved are made from the D delta 2 segment. The chromosome breakpoints are clustered within a region of approximately 263 base pairs of chromosome 10. The results suggest that the translocation of the TCR delta chain locus to a locus on 10q, which we have designated TCL3, results in deregulation of this putative oncogene, leading to acute T-cell leukemia.

Chromosomal translocation in T-cell leukemia line HUT 78 results in a MYC fusion transcript.

Primary cultures and established cell lines derived from human T-cell leukemias were analyzed for genomic rearrangements in the region 3' of the MYC locus. A T-cell leukemia line, HUT 78, whose 3' MYC region is rearranged, carries a chromosome t(2;8) juxtaposition; i.e., a locus derived from chromosome region 2q34 is attached to the 3' end of one MYC allele. The t(2;8) rearrangement in the HUT 78 cell line results in expression of a fused transcript encompassing the MYC gene and a locus designated TCL4 (T-cell leukemia/lymphoma 4), which normally resides on chromosome 2. The steady-state level of MYC-TCL4 fusion transcripts in HUT 78 cells is significantly higher than the MYC RNA level found in several other B- and T-cell lines. The production of fused MYC-TCL4 transcripts in a leukemic cell line raises the possibility that other B- and T-cell leukemias may express MYC fusion transcripts as an integral step in their pathogenesis.

Sequence analysis of the MYC oncogene involved in the t(8;14)(q24;q11) chromosome translocation in a human leukemia T-cell line indicates that putative regulatory regions are not altered.

We have cloned the translocation-associated and homologous normal MYC alleles from SKW-3, a leukemia T-cell line with the t(8;14)(q24;q11) translocation, and determined the sequence of the MYC oncogene first exon and flanking 5' putative regulatory regions. S1 nuclease protection experiments utilizing a MYC first exon probe demonstrated transcriptional deregulation of the MYC gene associated with the T-cell receptor alpha locus on the 8q+ chromosome of SKW-3 cells. Nucleotide sequence analysis of the translocation-associated (8q+) MYC allele identified a single base substitution within the upstream flanking region; the homologous nontranslocated allele contained an additional substitution and a two-base deletion. None of the deletions or substitutions localized to putative 5' regulatory regions. The MYC first exon sequence was germ line in both alleles. These results demonstrate that alterations within the putative 5' MYC regulatory regions are not necessarily involved in MYC deregulation in T-cell leukemias, and they show that juxtaposition of the T-cell receptor alpha locus to a germ-line MYC oncogene results in MYC deregulation.

A common mechanism of chromosomal translocation in T- and B-cell neoplasia.

The chromosomal breakpoint involved in the t(8;14)(q24;q11) chromosome translocation in the SKW-3 cell line, which directly involves the 3' flanking region of the c-myc gene, was cloned and sequenced. The breakpoint on chromosome 8 mapped to a position 3 kb 3' of c-myc while the chromosome 14 breakpoint occurred 36 kb 5' of the gene for the constant region of the alpha chain of the T-cell receptor (TCR). The translocation resulted in a precise rearrangement of sequences on chromosome 8 and what appears to be a functional J alpha segment on chromosome 14. Signal sequences for V-J joining occurred at the breakpoint positions on both chromosomes 14 and 8, suggesting that the translocation occurs during TCR gene rearrangement and that it is catalyzed by the enzymatic systems involved in V-J joining reactions. The involvement of c-myc in the translocation and the association of joining signals at the breakpoints provides a parallel to the situation observed in the translocations involving c-myc and the immunoglobulin loci in B-cell neoplasms and suggests that common mechanisms of translocation and oncogene deregulation are involved in B- and T-cell malignancies.

Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation.

From an acute B-cell leukemia cell line, a DNA probe was obtained that was specific for chromosome 18 and flanked the heavy chain joining region of the immunoglobulin heavy chain locus on chromosome 14. This probe detected rearrangement of the homologous DNA segment in the leukemic cells and in follicular lymphoma cells with the t(14:18) chromosome translocation but not in other neoplastic or normal B or T cells. The probe appears to identify bcl-2, a gene locus on chromosome 18 (band q21) that is unrelated to known oncogenes and may be important in the pathogenesis of B-cell neoplasms with this translocation.

Procedure for purification of Escherichia coli ribonucleic acid synthesis termination protein rho.

An improved purification procedure is described for the rho transcription termination factor of Escherichia coli. The method involves lysozyme--sodium deoxycholate lysis, Polymin P fractionation, and chromatography on phosphocellulose, poly(uridylic acid)--Sepharose, and AMP--agarose. The method yields up to 9 mg of electrophoretically pure protein from 200 200 g of E. coli MRE 600. From quantitative amino acid analysis rho is calculated to have an E280nm (1%) of 3.7 +/- 0.3. The purified rho has an ATPase specific activity of 32 nmol of Pi released min-1 microgram-1 when poly(cytidylic acid) is used as a cofactor, and it functions effectively in termination of T7 DNA transcription. A subunit molecular weight of 48 000 for rho was determined by phosphate-buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The amino acid composition and circular dichroism spectrum in the far-ultraviolet for rho are presented.