Mitchell R. Smith

A unique EBV-negative low-grade lymphoma line (WSU-FSCCL) exhibiting both t(14;18) and t(8;11)

A new human B-cell line, WSU-FSCCL, was established from the peripheral blood of a patient with low-grade follicular small cleaved cell lymphoma in leukemic phase. Both the fresh lymphoma cells and the established cell line exhibit t(14;18)(q32;q21) and t(8;11)(q24;q21) chromosomal translocations, 6q-, 1p+, and +i(1q). PCR analysis confirmed the juxtaposition of the major breakpoint-cluster region of bcl-2 with immunoglobulin heavy chain (JH) gene rearrangements. Southern analysis demonstrated that the 8q24 breakpoint was 5 of c-myc exon 1. The new line grows as a single-cell suspension with a doubling time of approximately 26 hours. It expresses cytoplasmic and cell surface IgM-kappa and reacts with monoclonal antibodies to B-cell antigens. Cells are negative for T-cell and myeloid/monocyte antigens as well as for Epstein-Barr virus nuclear antigen (EBNA). DNA histogram generated by flow cytometry indicated a near diploid stemline. While t(14;18) is common in follicular lymphomas, the t(8;11) is unusual in lymphomas, although it does involve a region frequently aberrant on chromosome 8. The rearrangement of c-myc may have conferred an aggressive clinical behavior seen in the terminal phase of the disease. The role of 11q21 remains undetermined.

A human B-cell lymphoma line with a de novo multidrug resistance phenotype

A human diffuse large cell lymphoma line (WSU‐DLCL) expressing multidrug resistance (MDR) was established from a patient with primary chemotherapy‐resistant disease. This cell line has the same phenotypic features as malignant cells from the patient. The established cell line has features of a mature B‐cell neoplasm with no evidence for commitment to other lineages. WSU‐DLCL grows in suspension forming relatively large clumps of cells with a doubling time of 20 hours. By light microscopic examination, the cells are very large with primitive lymphoid features, have a large amount of cytoplasm containing numerous vacuoles and an irregular outline. Immunophenotypic characterization by monoclonal antibodies and flow cytometric analysis showed a monoclonal IgM kappa B‐cell phenotype with high expression of the multidrug‐resistant P‐glycoprotein compared with either normal peripheral blood lymphocytes or cells of the REH cell line. The cells were negative for T‐cell and myeloid/monocyte antigens as well as Epstein‐Barr virus nuclear antigen (EBNA). In addition, the cell line expressed high levels of MDR RNA. DNA histogram generated by flow cytometry indicated a DNA index of 1.83. Cytogenetic analysis confirmed hypertriploidy and showed complex chromosomal abnormalities including 14q+. This cell line should be a valuable tool to study the role of the MDR gene in the primary resistance of lymphomas to chemotherapy and to facilitate therapeutic investigations. Cancer 1992; 69:1468‐1474.

Thymosin α1 does not promote growth or oncogenic transformation

Abstract Thymosin α1(Tα1) is an immune modulatory peptide which has been evaluated in a variety of clinical trials. Although no in vivo adverse effects, including enhancement of tumor growth, have been noted, in vitro studies suggesting a role for Tα1 in cell growth have been reported. The studies presented in this report evaluated both exogenously added Tα1 and endogenously expressed Tα1 as factors which could either promote growth of tumor cells or induce transformation. No effect of exogenous Tα1 on cell growth was found. NIH3T3 cells transfected with cDNA for the precursor ProThymosin α (ProTα) expressed elevated levels of authentic Tα1 but did not demonstrate either enhanced proliferation in liquid culture or transformation as defined by the loss of contact inhibition or anchorage independent growth in soft agar. Thus these studies argue against the hypothesis that Tα1 is either an intracellular or extracellular growth promoter.

Emergence of myeloid stem cell line from T-lymphoid blastic phase of chronic myeloid leukemia in culture

A new cell line designated JA-CML was derived from the peripheral blood of a patient with blastic phase CML. Sequential evolution of phenotypic and genetic markers was demonstrated during adaptation from primary to continuous culture in vitro. In the primary sample the majority of blast cells displayed the early T-cell markers, CD7, HLA-DR, and TdT, but were negative for the common ALL antigen (CALLA), CD4 and CD8. Simultaneously, unstimulated metaphase cells showed great karyotypic variation with a range of 43-46 chromosomes per cell. Clonal changes included the Ph chromosome t(9;22), loss of the Y and gain of several altered chromosomes. The cells grew slowly in suspension during the first 10 weeks of culture. During that time, cells still expressed the CD7 and HLA-DR antigens. Karyotypic analysis at ten weeks showed a pattern of 46,X,-Y,t(9;22),+8 in more than 90% of metaphases with disappearance of all other abnormal chromosomes noted in the original sample. A tetraploid subline exhibiting duplication of most chromosomes, including the Ph, comprised the remaining metaphases. Upon further cultivation in vitro, the cells transformed spontaneously over a period of several weeks, from T-lymphoid into myeloid cells. Expression of CD7 was lost, but reactivities with monoclonal antibodies to CD34, CD33 and CD13 were newly acquired. The karyotype was hypertriploid and all cells carried two copies of t(9;22) and lacked normal copies of No. 9 or Y. The cells have since maintained stable cytogenetic and phenotypic profiles. Molecular rearrangement of the breakpoint cluster region was identified in the primary blasts and the established line and T-cell receptor gene rearrangements were not found. These observations suggest that the leukemic blast arose from primitive stem cells, not irreversibly committed T cells, and that these stem cells retained the capacity to differentiate along the myeloid pathway.

Mutation of K234 and K236 in the voltage-dependent anion channel 1 impairs its insertion into the mitochondrial outer membrane.

Previous in vitro studies indicated that mutation of bothK234 and K236 to arginine, glutamine, or glutamic acid impaired the abilityof the voltage-dependent anion channel (VDAC1) to insert into the outermembrane of the mitochondria (Smith et al. 1995). These same mutantswere expressed in a strain of Saccharomyces cerevisiae with adisruption in the VDAC1 gene. The mutant VDAC1 forms were found in themitochondria suggesting that they were correctly sorted to the outermembrane. However, only very small amounts of the mutants were inserted intothe mitochondrial membranes. Mitochondria isolated from the strains expressingthe mutants were capable of catalyzing the translocation of both wild-typeVDAC1 and pre-alcohol dehydrogenase III indicating that the translocationapparatus was functional. These results confirm the previously drawnconclusion that K234 and K236 are part of a membrane insertion motif. Thefailure of the mutant VDAC1 forms to insert did not cause VDAC1 precursors toaccumulate in the soluble cell cytoplasm or in the microsomal fraction. Theapparent lack of a “precursor pool” suggested that apost-transcriptional control mechanism might limit the amounts of VDAC1precursors in the cell. Such a control mechanism is consistent with theobservation that the amount of VDAC1 was very similar after epichromosomal(gene in a 2u plasmid controlled by a Gal1 promoter) and chromosomalexpression (endogenous gene controlled by the endogenous promoter).