Telomere amount and length assay.

PURPOSE: Telomeres are specific DNA structure at the ends of chromosomes to protect chromosomes from fusion, recombination, and degradation. Telomere length changes are implicated in cell senescence, aging, tumorigenesis, and DNA repair. The standard method for measuring telomere length is Southern blot analysis. This method has several disadvantages, i.e., loss of DNA during membrane blotting, high background due to nonspecific binding of telomere probe to membrane, and loss of telomeric signal due to extensive washing. These limitations resulted in a low signal-to-noise ratio and, therefore, reduced sensitivity and reproducibility. The multi-step Southern blot is also highly labor-intensive. The present study was to develop a more quantitative assay of telomeric amount and length (TALA). METHODS: TALA was based on solution hybridization and did not require blotting, prehybridization, and washing. The major steps were (a) DNA preparation and digestion with restriction endonucleases, (b) hybridization between DNA and telomeric probe, (c) agarose gel electrophoresis, and (d) autoradiography and data analysis. RESULTS: The telomere amount measured by TALA was linearly correlated with the amount of DNA analyzed (r2 = 0.985, P < 0.01). The telomere length measured by TALA also correlated with the telomere length determined by fluorescence in situ hybridization (r2 = 0.99, P < 0.01). Compared to the Southern blot analysis, TALA showed a 4-fold greater sensitivity, 4.6-fold higher signal-to-noise ratio, >2 fold-higher reproducibility, and 4-fold less time requirement. CONCLUSION: We report here a rapid, sensitive, and quantitative assay for measuring telomere length and amount.

A non-characteristic response of L1210 cells to lovastatin.

Isoprenylated proteins are believed to play an important role in DNA synthesis and subsequent cell cycle progression. Inhibition of the biosynthesis of these isoprenylated proteins results in a decrease in DNA synthesis and a characteristic G1 blockade of the cell cycle. This inhibition can be achieved by incubation of cells in the presence of a hydroxy-methylglutaryl-coenzyme A reductase inhibitor (lovastatin) and can be reversed by addition of exogenous mevalonate. When incubated in the presence of lovastatin, L1210 wild-type cells are not inhibited at G1 but rather progress to a G2/M blockade, which is reversible with exogenous mevalonate. Thymidine incorporation has also shown that DNA synthesis is occurring until this blockade is achieved. However, when L1210 cells resistant to cisplatin are incubated in the presence of lovastatin, the characteristic G1 cell cycle blockade and DNA synthesis inhibition occur. The understanding of this mechanism and the role that isoprenylated proteins play in the regulation of DNA synthesis and cell cycle progression may gain great insight into the abnormal control of the cancer cell.

Nonclottable fibrin obtained from partially reduced fibrinogen: characterization and tissue plasminogen activator stimulation.

Out of 29 disulfide bonds in human fibrinogen, 7 were cleaved during limited reduction under nondenaturing conditions in calcium-free buffer: 2 A alpha 442Cys-A alpha 472Cys and 2 gamma 326Cys-gamma 339Cys intrachain disulfide bonds in the carboxy-terminal ends of the A alpha- and gamma-chains and the symmetrical disulfide bonds at gamma 8Cys, gamma 9Cys, and A alpha 28Cys. We studied the loss of thrombin clottability that followed limited reduction and the increase in the susceptibility of the fibrinogen A alpha 19-A alpha 20 bond to hydrolysis by thrombin. Using differential scanning calorimetry, we show that the extent of unfolding and denaturation of specific domains following limited reduction is small. Heat absorption peaks corresponding to the melting of the major regions of compact structure give high calorimetric enthalpies, as in untreated nonreduced fibrinogen, indicating that substantial regions of native structure are still present in partially reduced fibrinogen. Thrombin releases fibrinopeptide A at an identical rate as in nonreduced fibrinogen while fibrinopeptide B release is slower. Sedimentation velocity studies show that thrombin treatment leads to complex formation; however, gelation does not occur. Amino-terminal analysis indicates that the second thrombin cleavage in the A alpha-chain at A alpha 19-A alpha 20 takes place only after fibrinopeptide A release. Thus, the loss of clottability appears to result from perturbation of carboxy-terminal polymerization sites, probably a consequence of gamma 326Cys-gamma 339Cys intrachain disulfide bond cleavage. The thrombin-treated partially reduced fibrinogen remains soluble in buffered saline and fully expresses at least one epitope, B beta 15-21, unique to fibrin. Furthermore, this nonclottable form accelerates the tissue plasminogen activator dependent conversion of plasminogen to plasmin.