Tumors frequently develop resistance to cisplatin, a platinum drug used as a cornerstone of present-day chemotherapy regimens, significantly decreasing its usefulness in the clinic. Although it is known that cisplatin-resistant (CP-r) cancer cells commonly grow more slowly and exhibit reduced uptake of various compounds, including nutrients, the effect of tumor metabolism on cisplatin resistance is unclear. It was found that in CP-r cells, uptake of 2-deoxyglucose was reduced due to dysfunction and altered morphology of mitochondria compared with cisplatin-sensitive parental cancer cells. The CP-r cells overexpressed SIRT1, a histone deacetylase that plays a central role in DNA damage response and transcriptional silencing. Incubation of drug-sensitive cells in low glucose medium induced the expression of SIRT1 and increased cellular resistance to cisplatin. Reduced SIRT1 expression by a SIRT1 SMART small interfering RNA duplex sensitized the >20-fold resistant CP-r cells to cisplatin treatment 1.5-to 2-fold, and SIRT1 overexpression by SIRT1 cDNA transfection increased cisplatin resistance in cisplatin-sensitive cells by 2-to 3-fold. Our findings therefore suggest that reduced glucose use and altered mitochondrial metabolism mediated by SIRT1 is one of several alterations that contribute to cellular resistance

Two monoclonal antibodies (OKT27 and OKT27b) have been produced that react with distinct epitopes of a 95-kDa peptide. The T27 antigen is widely distributed, being expressed on B lymphocytes, monocytes, and adult Tleukemic cells but not on polymorphonuclear leukocytes or platelets. There was a low level of T27 expression on resting T cells that increased on T-cell activation. In preliminary studies, the OKT27b antibody coprecipitated a 55-kDa peptide, as well as the 95-kDa peptide, from the radiolabeled cells of the HuT 102B2 cell line. Preclearance with anti-Tac, a monoclonal antibody to the 55-kDa peptide of the multichain interleukin 2 receptor, removed the 55-kDa but not the 95-kDa peptide from subsequent OKT27b immunoprecipitates of HuT 102B2 extracts, suggesting the possibility that the T27 peptide was associated with the Tac peptide. However, the precipitation of the p55 Tac peptide by OKT27b was quite inconsistent. Thus, additional information was sought using a flow cytometric energy transfer technique to provide a physical estimation of the proximity between the Tac and the T27 peptides. The flow cytometric version of the fluorescence resonance energy transfer technique permits the determination of inter-and intramolecular distances at 2-to 10-nm levels on a cell-by-cell basis. Using this approach, there was a mean energy transfer of 7.3% with HuT 102B2 cells when fluorescein isothiocyanate anti-Tac served as the donor and tetramethylrhodamine isothiocyanate OKT27 served as the acceptor. In contrast, there was no energy transfer in comparable studies observed when fluorescein anti-Tac and rhodamine anti-transferrin receptor antibodies were used. These observations support the conclusion that there is a close nonrandom proximity in HuT 102B2 cells between the 95-kDa peptide identified by the OKT27 monoclonal antibody and the p55 Tac peptide of the multichain interleukin 2 receptor.T lymphocytes stimulated with antigen or mitogen produce interleukin 2 (IL-2) (1, 2). T-cell growth is dependent on the interaction of IL-2 with high-affinity IL-2 receptors that are not present on resting cells but are induced and expressed on T cells after activation (3-5). There are at least two classes of IL-2 receptors that differ markedly in their affinities for IL-2 (6). Both classes of receptors share the same Tac peptide (55 kDa) (4,5). To define the molecular basis for high-and low-affinity receptors and to determine the mechanism whereby IL-2 communicates a signal to the nucleus, we have investigated the possibility that the IL-2 receptor is a complex with multiple peptide chains in addition to the one identified by anti-Tac (7). Using crosslinking methodology, we have identified a non-Tac IL-2 binding peptide of 75 kDa and have proposed that the 75-kDa IL-2 binding peptide is associated with the 55-kDa Tac peptide to form the highaffinity IL-2 receptor complex (7). Recently, two additional monoclonal antibodies, OKT27 and OKT27b, were developed in an effort to identify distinct human T-cell activation antig...

vs. pH profile (zero buffer) in Figure 2 in the pH region from 7.5 to 8.0, i.e., that for rate-limiting attack of OH" on the oxocarbonium ion.At pH >10 the ring opening reaction becomes independent of pH. Similar pH-independent reactions have been found in .the hydrolysis of acetals and ketals subject to general acid catalysis.7,8•10 These reactions involve rate-determining unimolecular C-O bond breaking.8 Thus the analogous reaction of II may also involve a unimolecular decomposition (VIII). Such a reaction would be 8-/VQ, z=\ /H-\_J~N(CH3>2 VIII due to the significant oxocarbonium ion stabilization and the ease of C-O bond breaking brought about by relief of steric strain in the acetal of trans-1,2-cyclohexanediol. An alternative possibility is that water is acting as a general acid, which would require a second-order rate constant of 1.1 X "6 M"1 s'1 (kj55.5 M). This second-order rate constant fits reasonably well on a Bronsted plot of slope -0.7 including points for the general acids and hydronium ion.In summary, in the plot of log kobsd vs. pH for the hydrolysis of trans-1,2-cyclohexanediol p-(dimethylamino)benzylidene acetal in H20 there are seven inflections, only one of which is due to an ionizable group (the p-dimethylamino group conjugate acid). As discussed, the other inflections are produced by two changes in rate-determining step and four changes in mechanism as pH is increased. This novel situation comes about because of steric strain in the acetal, so that ring opening is rapid, and the cyclic structure of the acetal, which can lead to reversibility of ring opening. The resultant compromise between these opposing factors is one in which each step becomes rate limiting in turn as pH is increased. Hemiacetal breakdown can only be rate determining at pH <6 because of OH" catalysis at higher pH. Rate-limiting hydrolysis of the protonated and neutral species of the hemiacetal at low pH is brought about because of the rapid hydronium ion catalyzed ring opening or L•2aH > k2. As the pH is increased above 6 attack of a water molecule on the oxocarbonium ion intermediate becomes rate limiting because at those pH values the hydroxide ion catalyzed breakdown of the hemiacetal is rapid with fc3(OH~) > fc-2flH• The mechanism changes at pH >7 to attack of OH" on the oxocarbonium ion, and as the concentration of OH" becomes larger the hydronium ion catalyzed ring opening becomes rate determining, i.e., &oh(OH~) > The ring-opening is also catalyzed by general acids. Finally at pH >10 the mechanism changes to pH-independent rate-determining ring opening. Thus, alt of the mechanisms and rate-determining steps for the hydrolysis of an acetal are represented on one remarkable log kobsd vs. pH profile (Figure 2).

No abstract