Flavopiridol induces apoptosis and caspase-3 activation of a newly characterized Burkitt's lymphoma cell line containing mutant p53 genes.

Burkitt's lymphoma cell lines have been important in vitro models for studying the pathogenesis of Burkitt's lymphoma (BL) and for exploring new treatment strategies. A new EBV(-) Burkitt's lymphoma cell line (GA-10) was established from a patient with a clinically aggressive, chemorefractory BL and characterized. Although functional p-glycoprotein could not be demonstrated by dye-efflux assays, both p53 genes were mutated in the GA-10 cells, perhaps contributing to the resistant phenotype of the original neoplasm. Two properties of BL cells which may be useful targets for novel cytotoxic therapeutics are their surface expression of CD77, the receptor for Shiga toxin (Stx), and their high rate of proliferation. Expression of CD77 on the GA-10 cells was heterogeneous in that certain subclones expressed high levels of CD77 and correspondingly exhibited strong growth inhibition by Stx while others showed low levels of CD77 expression and weak Stx-induced growth inhibition. Flavopiridol, a potent inhibitor of cell cycle progression through G1 and G2, induced cytotoxicity of the GA-10 cells with an LC(50) of approximately 40 nM vs 70 nM for HL-60 cells (P < 0.05). The concentrations of flavopiridol at which only 10% of the cells were viable (LC(10)) were approximately 280 nM for the GA-10 cells and 520 nM for the HL-60 cells (P < 0.05). Dose-related induction of apoptosis in response to flavopiridol was demonstrated in the GA-10 cells by morphology, TUNEL assay, and activation of caspase-3. Flavopiridol was also cytotoxic to seven other BL cell lines tested. These data suggest that flavopiridol may have therapeutic value in the treatment of Burkitt's lymphoma.

PBK/TOPK is a novel mitotic kinase which is upregulated in Burkitt's lymphoma and other highly proliferative malignant cells.

PBK/TOPK is a recently cloned serine/threonine kinase which is phosphorylated during mitosis. Earlier work indicated that this kinase is upregulated in a Burkitt's lymphoma cell line (GA-10). To determine whether PBK/TOPK is upregulated in other mitotically active neoplastic cell lines and tissues, Northern analysis was performed on a panel of malignant cell lines and on clinical samples from patients with leukemia or lymphoma. While PBK/TOPK mRNA was not detectable in normal peripheral blood cells and was weakly expressed in hyperplastic tonsillar B-cells, significantly higher levels of mRNA were detected in 8 Burkitt's lymphoma cell lines, 10 other neoplastic cell lines, and 2 clinical samples-one derived from a patient with ALL and a second derived from a patient with relapsed myeloma. In addition, Northern analysis of fetal tissues showed upregulated expression of PBK/TOPK in fetal kidney, lung, spleen, brain, and testis. These data suggest that PBK/TOPK expression is increased in highly proliferative malignant cells and during normal fetal development.

Characterization of the mouse Nktr gene and promoter.

We have cloned and determined the structure of the 5' region of the mouse Nktr gene located on the distal end of mouse chromosome 9. This gene encodes an NK-cell-specific 150-kDa protein (NK-TR) homologous to cyclophilin, Nopp140, and SR-containing proteins. NK-TR expression is important for maintaining the lytic activity of natural killer cells. The region of the Nktr gene cloned in this study spans 25 kb and contains the promoter, eight exons, and a single alternative exon. The boundaries of exons 6-8 and the alternate splicing events in this region are identical to those previously described for the human NKTR gene. The Nktr promoter region has features that are typical of a housekeeping gene, including high G + C content, high frequency of CpG dinucleotides, and a lack of canonical TATAA and CCAAT boxes. The activity of Nktr promoter/beta-gal reporter constructs was equivalent in lymphocyte and fibroblast cell lines, suggesting that NK-TR protein expression is regulated by posttranscriptional mechanisms. In support of this hypothesis, two levels of splicing control have been identified within the Nktr gene. A 10-kb intron was found to remain in mRNAs produced in bone marrow, and an alternative exon capable of interrupting the Nktr open reading frame was found in immature NK cells. A conserved intronic sequence has been identified that may be important for the regulation of the Nktr gene by pre-mRNA splicing.