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A Bakhshi

Publications and source records attributed to A Bakhshi.

At least 19 recordsLinked to original sources
PubMed1990

Mechanism of bcl-2 activation in human follicular lymphoma.

The t(14; 18) chromosomal translocation of human follicular lymphoma recombines the bcl-2 gene from chromosome 18 with the immunoglobulin heavy chain joining region. In the t(14; 18) translocation bearing cell line SU-DHL-6, this results not only in an inappropriately high rate of bcl-2 transcription for a mature B cell, but also in two potentially critical point mutations. To determine the relative importance of these mutations, we searched for their presence in DNA from the involved lymph nodes of 12 patients with t(14; 18) follicular lymphoma. bcl-2 genomic sequences were specifically amplified by the polymerase chain reaction technique and then directly sequenced. None of the 12 samples analysed revealed the codon 7 or codon 129 mutation detected in SU-DHL-6. We conclude that abnormal expression of bcl-2 rather than structural alterations at codon 7 or 129 play an important role in the disordered growth and differentiation of follicular B-cell lymphoma.

B-Lymphocytes↗
PubMed1988

Gene mutations and alternate RNA splicing result in truncated Ig L chains in human gamma H chain disease.

The lack of covalently associated L chains features H chain disease proteins produced in some human B cell lymphoproliferative disorders. We cloned and characterized the single rearranged kappa L chain gene from the leukemic lymphocytes of a patient (RIV) affected with gamma 1 H chain disease, to determine the molecular basis for absent L chain. This kappa allele had undergone an effective V-J rearrangement. Extensive somatic mutation focused about the V-J region created a sequence that was only 75% homologous to its germ-line counterpart. Altered acceptor (V kappa) and donor (J kappa) splice sites resulted in an aberrant splice between the leader and C kappa exons and a truncated 850-bp kappa mRNA. RIV leukemic cells as well as myeloma cells transfected with the RIV kappa gene synthesized a truncated protein. Simultaneous defects in H and L chains genes may reflect a hypermutational mechanism for Ig genes in B cells.

Amino Acid Sequence↗
PubMed1988

Multiple genomic defects result in an alternative RNA splice creating a human gamma H chain disease protein.

Heavy chain diseases (HCD) are human lymphoproliferative disorders in which a clonal B cell population produces Ig molecules made of truncated H chains without associated L chain. We characterized the rearranged H chain gene and its mRNA from the leukemic cells of a patient (RIV) with gamma-HCD. The abnormal RIV serum Ig consisted of shortened, dimeric gamma 1-chains which had an amino terminus within the hinge region. RIV lymphoblasts possessed a foreshortened (1200 bp) gamma 1-mRNA which had sequences for only the leader, hinge, second, and third constant region domains (CH2 + CH3), but lacked variable (VH) and CH1 information. Sequence of the productive gamma 1 allele revealed it had undergone VH-JH and H chain class switch recombinations. However, normal RNA splice sites had been eliminated by a DNA insertion/deletion (VH acceptor site), mutations (JH donor site), or a large deletion (CH1 region). Inserted sequences were of non-Ig and apparently non-genomic origin. These DNA alterations resulted in aberrant mRNA processing in which the leader region was spliced directly to the hinge region, accounting for the HCD protein.

Chromosome Aberrations↗
PubMed1988

Consequences of the t(14;18) chromosomal translocation in follicular lymphoma: deregulated expression of a chimeric and mutated BCL-2 gene.

The t(14;18) chromosomal translocation of human follicular lymphoma recombines the candidate transforming gene bcl-2, located at 18q21, with the immunoglobulin (Ig) H-chain joining region (JH) at 14q32. To elucidate the consequences of this translocation, we cloned bcl-2 cDNAs from a pre-B cell line (Nall-1) and a t(14;18) lymphoma cell line (SU-DHL-6) and compared these sequences with their genomic counterparts. These studies revealed the complexity of bcl-2 gene expression in which six potential polyadenylation signals in exon 3 and two different 5' exons (exons 1 and 2) and promoters are alternatively used to generate different sized bcl-2 mRNAs. A single open reading frame (ORF), at the junction of exons 2 and 3, predicts a 239 amino acid, 26 kD protein. Most chromosome 18 breakpoints cluster within a 150 bp region of exon 3. In SU-DHL-6 the t(14;18) translocation juxtaposes a truncated bcl-2 gene with J6 in a tail-to-head configuration, resulting in the deregulated expression of chimeric bcl-2/Ig transcripts. Importantly, the SU-DHL-6 bcl-2 cDNA also contained several point mutations in the ORF, two of which altered the primary amino acid sequence. The deregulated expression of an altered bcl-2 gene may play a critical role in the disordered growth and differentiation of follicular B cell lymphoma.

Amino Acid Sequence↗
PubMed1987

Clonal evolution of t(14;18) follicular lymphomas demonstrated by immunoglobulin genes and the 18q21 major breakpoint region.

A 2.8-kilobase major breakpoint region on chromosome segment 18q21 is the site of most t(14;18) translocations typical of human follicular lymphomas. Breaks are focused at the 5' end of joining (JH) regions of immunoglobulin (Ig) on chromosome 14, indicating that the translocation occurs at a pre-B-cell stage during attempted heavy (H) chain joining. A new gene from 18q21 (Bcl-2) is placed in the H chain locus creating a unique, translocation-specific JH;18q21 rearrangement that presumably represents a transformation event. In addition, normal Ig gene joining occurs in a H before light (L) chain and K before lambda cascade, creating ordered clonal markers. These serial markers were examined to determine if variations in Ig gene patterns during the natural history of lymphomas represent the emergence of truly separate neoplasms or heterogeneity of a single neoplasm. We examined 45 serial biopsies from 16 B follicular lymphoma patients; six cases showed variation in Ig gene patterns over time. Seven individuals had a detectable JH;18q21 rearrangement present, and it remained unchanged over 5-10 years. Further rearrangements of H chain genes occurred on the normal chromosome 14 within evolving subclones of the original tumor. Lambda L chains also underwent additional rearrangements in two instances, while K gene patterns remained unchanged. All variations in the normal H and L chain genes were 2 degrees rearrangements occurring at a mature B-cell stage following the initial successful rearrangement of a H and L chain. In contrast the t(14;18) breakpoint was conserved in each individual, indicating that evolving neoplastic subpopulations arose from a common clonal progenitor cell.

Alleles↗
PubMed1987

Mechanism of the t(14;18) chromosomal translocation: structural analysis of both derivative 14 and 18 reciprocal partners.

To elucidate the mechanism of the t(14;18)(q32;q21) chromosomal translocation found in follicular lymphoma, we examined the structure of both derivative (der) chromosomal breakpoints as well as their germ-line predecessors. We noted that chromosome segment 18q21 was juxtaposed with immunoglobulin heavy (H) chain gene diversity (DH) regions on all five der(18) chromosomes we examined, and we confirmed the juncture with immunoglobulin H-chain gene joining (JH) regions on the der(14) chromosome. However, the t(14;18) was not fully reciprocal in that chromosome 14 DNA between the DH and JH regions was deleted. Furthermore, extra nucleotides, reminiscent of "N" segments, were present at the der(14) and possibly der(18) junctions. This indicates that despite the mature B-cell phenotype of follicular lymphoma, the t(14;18) occurs during attempted DH-JH joining, the earliest event in immunoglobulin rearrangement in a pre-B-cell. Our detailed analysis of the germ-line 18q21 region indicated that most breakpoints clustered within a 150-base-pair major breakpoint region. However, we found no evidence for evolutionarily conserved immunoglobulin-like recombinational signals at 18q21, arguing against a role for immunoglobulin recombinase in chromosome 18 breakage. Instead, a direct repeat duplication of chromosome 18 sequences was discovered at both chromosomal junctures, typical of the repair of a naturally occurring staggered double-stranded DNA break. These results prompt a translocation model with illegitimate pairing of a staggered double-stranded DNA break at 18q21 and an immunoglobulin endonuclease-mediated break at 14q32 and with N-segment addition, repair, and ligation to generate der(14) and der(18) chromosomes.

Base Sequence↗
PubMed1987

Gene rearrangements as markers of clonal variation and minimal residual disease in acute lymphoblastic leukemia.

Immunoglobulin (Ig) heavy (H) and light (L) chain gene rearrangements were used as molecular markers of clonal evolution and minimal residual disease in B cell precursor acute lymphoblastic leukemia (ALL). All leukemic episodes within individual patients shared at least one identical Ig rearrangement and thus arose from a common clonal progenitor cell. Nine of 11 patients displayed completely identical patterns between leukemic episodes, while two of 11 patients demonstrated genetic progression between diagnosis and relapse as evidenced by additional rearrangements. These genetic changes marked the emergence of leukemic subclones. Ig gene rearrangements were also used as sensitive markers to identify clonal cell populations in ALL patients following induction or reinduction therapy and to search for residual bone marrow disease in patients in clinical remission or with isolated extramedullary relapse. DNA rearrangements provide tumor-specific markers to follow the genetic variation of ALL and may facilitate the early detection of recurrent disease.

B-Lymphocytes↗
PubMed1987

Refinement of lymphoma cytogenetics by the chromosome 18q21 major breakpoint region.

A small (2.8-kilobase, kb) major breakpoint region localized to segment 18q21 rearranges in greater than 70% of t(14;18)(q32;q21) lymphomas. This rearrangement interrupts the Bcl-2 gene and introduces it into the Ig locus at 14q32. The rearrangement between the joining region (JH) of Ig on chromosome 14 and the 18q21 region creates a translocation-specific DNA rearrangement. We generated probes that distinguish the 14;18 juncture on the derivative (der) 14 and der (18) chromosomes, providing a molecular approach to t(14;18) identification. Approximately 60% of unselected follicular lymphomas, 20% of diffuse large cell lymphomas, and 50% of adult undifferentiated non-Burkitt lymphomas demonstrated 14;18 rearrangements within the major breakpoint region. Examination of DNA for 14;18 rearrangements resolved the identity of 14q+ chromosomes in two patient's cells that lacked an obvious reciprocal partner. Identification of the exact restriction fragments that mediate translocations complements routine cytogenetics. The detection of DNA rearrangements does not require dividing cells or the presence of an identifiable reciprocal partner and can detect clonal translocation rearrangements when the neoplastic cells are only a minority of all cells present.

Chromosome Banding↗
PubMed1986

A pre-translational defect in a case of human mu heavy chain disease.

A patient (BW) was studied with Mu heavy chain disease (mu HCD) in whom a leukemic B-cell clone secreted a shortened monoclonal mu chain without associated light chain. The cells did, however, produce a normal-sized kappa light chain that was detected as urinary Bence-Jones protein. The cytoplasmic and secreted monomeric mu chain had an approximate mol. wt of 58,000. Radiochemical sequence analysis of the biosynthetically labelled mu chain revealed a protein that lacked the entire variable region. The sequence initiated at amino acid position 5 within the first constant region domain (CH1) of C mu. The primary in vitro translation product, the cytoplasmic and secreted proteins were all similarly truncated, thereby excluding extensive postsynthetic degradation. The mu RNA, that directed the synthesis of the truncated mu protein, was about 350 bp smaller than the normal mu RNA. Furthermore, by primer extension analysis it was possible to localize this deletion in the mu RNA to a region 5' of CH1. Thus, a defect at the level of Ig gene structure/assembly that deletes coding information or results in aberrant RNA processing must be responsible for the truncated mu HCD protein BW.

Amino Acid Sequence↗
PubMed1986

A DNA insertion/deletion necessitates an aberrant RNA splice accounting for a mu heavy chain disease protein.

The human heavy chain disease protein BW is an immunoglobulin mu-chain variant whose amino terminus is initiated at the fifth amino acid of the first constant region domain. We cloned and analyzed both rearranged heavy chain alleles from BW leukemic cells to determine the molecular basis for this deleted protein. The phenotypically excluded heavy-chain allele possessed two intermediate recombinations of separate variable-diversity (V-D) and diversity-joining (D-J) junctions, neither of which were expressed. The productive allele, responsible for the mu chain, had a complete V-D-J4 recombination but as a result of a single-base deletion possessed stop codons within the variable region. More important, a small DNA insertion/deletion eliminated the J4 donor splice site. This necessitated an aberrant RNA splice between the leader region and the first constant region domain creating a shortened 2.35-kilobase muRNA. A recognition sequence for signal peptidase predicted a cleavage at the fifth amino acid of the first constant region domain. These molecular events are responsible for the truncated mu chain that lacks a variable region and fails to assemble light chains.

Alleles↗
PubMed1986

Immunoglobulin chain gene rearrangements in a t(4;11) acute leukaemia with monocytoid blasts.

We report a case of acute leukaemia with the t(4;11) chromosomal translocation which, at initial diagnosis, had L-1 lymphoblasts that were positive for terminal deoxynucleotidyl transferase (TdT) and HLA-DR but negative for myeloid cytochemical markers. At last relapse the patient had mostly monocytoid blasts which were not TdT negative but were positive for HLA-DR, weakly positive for Sudan Black B (SB), periodic acid Schiff's (PAS), naphthol AS-D acetate esterase (NSE), chloroacetate esterase (CAE) and negative for acid phosphatase (AP) and nitroblue tetrazolium (NBT) reduction. Treatment with 12-o-tetradecanoylphorbol-13-acetate (TPA) in vitro induced differentiation to macrophage-like cells that were strongly positive for SB, PAS, NSE, AP, CAE and NBT reduction, indicating a latent monocyte-like phenotype. Thus the leukaemic cell clone or a precursor clone with the t(4;11) translocation manifested a lymphoid phenotype at initial diagnosis and a monocytoid phenotype at relapse. Immunoglobulin gene analysis of the monocytoid relapse blasts revealed rearrangements of the heavy chain gene alleles and germline light chain genes. Thus, the leukaemia clone with the t(4;11) chromosomal translocation could be a bipotential cell with heavy chain gene rearrangements occurring in a primitive cell which may retain the ability to differentiate along the myeloid-monocytoid lineage in response to the appropriate stimulus. Alternatively, these characteristics may result from a transformation associated event.

Cell Differentiation↗
PubMed1985

Cloning the chromosomal breakpoint of t(14;18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18.

Specific chromosomal translocations found in distinct neoplasms suggest that genes that flank such breakpoints play a critical role in transformation. We have characterized the t(14;18)(q32;q21) chromosomal translocation present in over 60% of human follicular lymphomas. We exploited an unexpected rearrangement of an Ig heavy-chain gene to clone the chromosomal breakpoint. An element isolated from 18q21 mediated translocations in all four t(14;18) bearing cell lines and in six of 11 follicular lymphomas, but did not normally rearrange in other B or non-B cells. The breakpoints clustered within a small 4.3 kb region on chromosome 18. The breakpoints on chromosome 14 were focused within or immediately 5' to JH. These breakpoints retained the Ig enhancer region close to a new transcriptional unit identified on chromosome segment 18q21. Since none of the cellular oncogenes are known to map to 18q21, cloning this element provides an opportunity to characterize a potentially new transforming gene.

Cell Line↗
PubMed1985

NIH conference. Molecular genetic analysis of human lymphoid neoplasms. Immunoglobulin genes and the c-myc oncogene.

Immunoglobulin genes responsible for individual antibodies are organized as discontinuous DNA segments in their germline form. As an uncommitted stem cell develops into an antibody-synthesizing plasma cell, rearrangements of these immunoglobulin gene segments serve to activate the genes and to generate the virtually unlimited capacity to synthesize antibodies that recognize potential antigens. The analysis of immunoglobulin gene structure and arrangement has been of immense value in the study of human lymphoid neoplasms. Recombinant DNA technology involving analysis of immunoglobulin gene arrangement has been used to classify neoplasms of previously uncertain lineage, aid in the diagnosis of neoplasms of the B-cell series, and define the state of differentiation of neoplastic B-cell precursors. Furthermore, the demonstration of translocation of a particular transforming gene, the c-myc oncogene, into the immunoglobulin gene locus in Burkitt's lymphoma has provided a major insight into the cause of malignant transformation of these lymphoid cells.

Alleles↗
PubMed1984

In vitro enhancement of immunoglobulin gene expression in chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (CLL) cells appear to be arrested in their differentiation so that little immunoglobulin is secreted in most cases. To determine their capacity for further differentiation we stimulated cells from a series of 10 cases of CLL with a phorbol ester and assayed for production of immunoglobulin protein, accumulation of immunoglobulin mRNA, and alterations in cell surface markers. We found that cells from all cases were induced to secret monoclonal immunoglobulin of the same heavy and light chain type as the surface membrane immunoglobulin type. Immunoglobulin secretion was preceded by a rapid increase in the levels of mRNA coding for IgM, predominantly the secretory form, mu s-mRNA, rather than the membrane form, mu m-mRNA. A similar selection of mu s- over mu m-mRNA is known to occur in plasma cells by a mechanism of differential processing of mRNA from a single mu-chain gene. Except for a decline in the expression of surface IgD, cell surface determinants remained unaffected both in terms of the percentage of positive cells and the relative number of sites per cell. In contrast to previous studies, these results indicate that CLL cells consistently retain the capacity to further differentiate toward plasma cells and secrete immunoglobulin. The immunoglobulin secretion is mediated, at least in part, by a developmentally regulated increment in mu s-mRNA.

B-Lymphocytes↗
PubMed1983

Immunoglobulin-gene rearrangements as unique clonal markers in human lymphoid neoplasms.

Immunoglobulin genes in their germ-line form are separated DNA subsegments that must be joined by means of recombinations during B-cell development. Individual immunoglobulin-gene rearrangements are specific for a given B cell and its progeny. We show that the detection of such gene rearrangements by Southern hybridization provides a sensitive marker for both clonality and B-cell lineage within lymphoid tissues lacking expression of definitive surface phenotypes. We have used these genetic markers in three ways: to establish a diagnosis of lymphoma in a neoplastic disorder of uncertain cell type, to show that some lymphomas that were previously classified as being of T-cell type in fact contain monoclonal B cells, and to detect clonal B-cell populations within lymphomatous tissues of uncertain immunotype and within an atypical lymphofollicular hyperplasia having no other clonal surface markers. These sensitive and unique indicators of clonality located directly at the DNA level are capable of providing insights into the cellular origin, early detection, and natural history of neoplasia.

B-Lymphocytes↗
PubMed1983

Lymphoid blast crises of chronic myelogenous leukemia represent stages in the development of B-cell precursors.

The origin and stage of differentiation of the blast-crisis cells in chronic myelogenous leukemia have remained uncertain. Because immunoglobulin heavy-chain and light-chain genes must undergo a DNA rearrangement during B-cell development but rarely do so in human non-B-cell lineages, we examined these genes in 18 episodes of chronic myelogenous leukemia. In eight of nine episodes of lymphoid blast crisis, heavy-chain genes were rearranged, and in three, rearrangements in light-chain genes were also present. In contrast, cells from chronic myeloid, myeloid blast, and erythroid-like phases retained germ-like immunoglobulin genes. The observed phenotypic markers and gene configurations revealed that most lymphoid blast crises represent stages of development of B-cell precursors. In two separate episodes of lymphoid crisis, cells from a single patient possessed identical heavy-chain but different light-chain-gene configurations. Thus, the precursor cells that monoclonally expand to produce a lymphoid crisis are capable of immunoglobulin-gene rearrangements and represent discrete steps in early B-cell maturation.

Antigens, Surface↗
PubMed1983

Rearrangement and expression of immunoglobulin genes and expression of Tac antigen in hairy cell leukemia.

The origin and exact stage of differentiation of the neoplastic cells that comprise hairy cell leukemia have remained uncertain. As Ig heavy and light chain genes must both undergo a DNA rearrangement during B-cell development but rarely do so within other hematopoietic lineages, we examined these genes in this leukemia. The neoplastic cells of all eight cases demonstrated rearranged heavy and light chain genes and, in two cases examined, contained the corresponding mRNA for heavy and light chain Ig. Consistent with this B-cell genotype, all cases displayed cell surface HLA-DR and B-cell-associated antigens. Unexpectedly, all cases demonstrated cell surface Tac antigen, which previously had been restricted predominantly to select T-cell malignancies and activated T cells. Prior studies suggested that the anti-Tac monoclonal antibody recognized a peptide associated with the binding of interleukin 2 (T-cell growth factor) in such T cells. Immunoprecipitation with anti-Tac and NaDodSO4/polyacrylamide gel electrophoresis revealed an antigen on leukemic hairy cells with a Mr of 53,000-57,000, identical in size to the receptor on activated T cells. This apparent biphenotypic status might reflect a transformation-associated expression of the Tac antigen in this leukemia. Alternatively, hairy cell leukemia may be a malignancy of a unique stage of normal B-cell differentiation in which the Tac antigen is expressed.

Antibodies, Monoclonal↗