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The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic progenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity.CD34(+) cord blood (CB) cells were cultured in serum-free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 14 days, the primitive functions of expanded and nonexpanded cells were determined in vitro using clonogenic cell (colony-forming cells, long-term culture initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis assays (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells also was assessed by determining their telomere length and telomerase activity. Levels of expansion were up to 1,613-fold for total cells, 278-fold for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 21-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they were able to generate myeloid progenitors and lymphoid cells for 5 months. These primitive progenitors engrafted the NOD-SCID bone marrow, which contained LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CDl9(-) cells sorted from the engrafted marrow were able to generate CD19(+) B-cells, CD56(+)CD3(-) NK cells, and CD4(+)CD8(+)alphabetaTCR(+) T-cells in specific cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 weeks of culture. These experiments provide strong evidence that expanded CD34(+) CB cells retain their ability to support long-term hematopoiesis, as shown by their engraftment in the NOD-SCID model, and to undergo multilineage differentiation along all myeloid and the B-, NK, and T-lymphoid pathways. The expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical applications.

The CDK10/PISSLRE gene has been shown to encode two different CDK-like putative kinases. The function(s) of the gene products are unknown, although a role at the G2/M transition has been suggested. We characterised two novel cDNAs. CDK10 mRNA quantity was not found to be correlated with cell proliferation status in HeLa or WI38 cell cultures or in human tissues. Relative levels of the four CDK10 isoforms were studied by RT-PCR, of which three were principally expressed. The two initially cloned isoforms predominated in human tissues, except in brain and muscle. Relative isoform levels did not vary during the cell cycle in culture, except when cells entered into the cell cycle. Finally, the predominant isoforms were shown to have different translation initiation sites and to have different subcellular distribution, due to an alternatively spliced nuclear localisation signal.

Stabilization of the topoisomerase-cleavable complexes is the common initial event leading to the cytotoxicity of topoisomerase I and II (top1 and top2) inhibitors. Using saintopin (STP), a poison of both topoisomerases, we studied top1- and top2-cleavable complexes (Yamashita, Y., Kawada, S.-Z., Fujii, N., and Nakano, H. (1991) Biochemistry 30, 5838-5845). top1 and top2 sites induced in the presence of STP showed the same preferences for the base located 3' to the topoisomerase-induced DNA break (position +1): preference for G and not C. A camptothecin-resistant top1 with a mutation (Asn722-->Ser) next to the catalytic tyrosine (Tyr723) was cross-resistant to STP, suggesting that both STP and camptothecin interact with the protein near the catalytic tyrosine. These results are consistent with a dual interaction of the drug with the enzyme and the DNA and provide further evidence for the "drug-stacking" model. This model proposes that topoisomerase inhibitors bind, possibly through hydrogen bonding and/or stacking, with one of the bases flanking the DNA termini (guanine at position +1 in the case of STP) and within the enzyme catalytic pocket, most likely by stacking with the catalytic tyrosine.

The non DNA intercalator streptonigrin was shown to inhibit topoisomerase II by stabilizing cleavable complexes (Yamashita et al, Cancer Res. 1990, 50, 5841). Streptonigrin-induced topoisomerase II cleavage sites were mapped in the c-myc proto-oncogene DNA. Streptonigrin induced a unique cleavage pattern. Its cleavage sites were less frequent than those induced by other topoisomerase II inhibitors. Strongly preferred bases were found in the middle of topoisomerase II DNA stagger, with thymine at position +2 and adenine at position +3, position +1 being the nucleotide covalently linked to topoisomerase II. Preference for bases not immediately flanking the cleavage sites has not been reported previously and indicates that a mechanism other than "drug stacking" within the DNA break is taking place with streptonigrin to stabilize cleavable complexes. An alternative model taking into account the unusual DNA binding properties of streptonigrin is proposed.

The inhibition of HIV-1 integrase by flavones and related compounds was investigated biochemically and by means of structure-activity relationships. Purified enzyme and synthetic oligonucleotides were used to assay for three reactions catalysed by integrase: (1) processing of 3' termini by cleavage of the terminal dinucleotide; (2) strand transfer, which models the integration step; and (3) "disintegration," which models the reversal of the strand transfer reaction. Inhibitions of all three reactions by flavones generally occurred in parallel, but caffeic acid phenethyl ester (CAPE) appeared to inhibit reaction 2 selectively. CAPE, however, inhibited reactions 1 and 3 effectively when preincubated with the enzyme, suggesting that this compound differs from the flavones primarily in requiring more time to block the enzyme. The core integrase fragment consisting of amino acids 50-212 retained the ability to catalyse reaction 3, and flavones and CAPE retained the ability to inhibit. Hence, the putative zinc-finger region that is deleted in this fragment is probably not the target of inhibition. Inhibition by flavones usually required the presence of at least one ortho pair of phenolic hydroxyl groups and at least one or two additional hydroxyl groups. Potency was enhanced by the presence of additional hydroxyl groups, especially when present in ortho pairs or in adjacent groups of three. Inhibitory activity was reduced or eliminated by methoxy or glycosidic substitutions or by saturation of the 2,3 double bond. These structure-activity findings for flavones were generally concordant with those previously reported for reverse transcriptase and topoisomerase II. These findings are discussed in the context of a review of the effects of flavones on various enzymes, the possible mechanisms of inhibition, and the potential for building upon a general pharmacophore to generate target specificity.