Fig. 3. Effect of BVDU combinatorial treatment on protein expression. A, Western blot analysis, expression of STAT3, JUN-D, and caspase-3 (proteolytically activated form) in response to cytostatic drug treatment. The expression levels were determined densitometrically (r. = recovery, see Fig. 1C). B, NQO1 enzyme activity (mean value ± SD, six independent experiments).

  We investigated NQO1 activity in cell extracts (16) after treatment with the cytostatic drugs ± BVDU (Fig. 3B). BVDU cotreated cells showed higher NQO1 activity than untreated control cells or cells treated with cytostatic drugs only. Interestingly, cells treated with MMC+BVDU, which caused the strongest antiproliferative effect, did not enhance NQO1 activity. To elucidate the effects of BVDU, we performed a two-dimensional gel electrophoresis and identified differentially expressed proteins by MALDI-MS (Table 1). During combinatorial MMC+BVDU treatment, or during recovery (MMC omitted, BVDU present) from combinatorial treatment, the expression of three major "clusters" of protein classes was affected: (a) microfilamental (or regulatory) proteins were up-regulated during recovery (actins, tubulin, myosin, and tropo-modulin); (b) proteins involved in ATP generation were down-regulated (succinate dehydrogenase, pyruvate dehydrogenase, and malic enzyme; however, malate dehydrogenase was up-regulated); and (c) proteins regulating DNA repair were suppressed (APEX and UBE2N). One protein with oncogenic potential, DDX1, was affected by BVDU alone. In total, 75% of the spots were identified by MALDI-MS.

Table 1 Effects of cotreatment of MMC with BVDU on protein expression 

Proteins Fold change BVDU only vs. control control (=1) Dead/H Box 1; DDX1 -2.65 MMC + BVDU vs. MMC only MMC only (= 1) Protein disulfide isomerase/oxidoreductase; PDI +2.56 Malate dehydrogenase, soluble; MDH1 +3.25 Myosin, heavy chain 1, neuronal similarity, adult; MYH1 +3.23 Ubiquitin-conjugating enzyme E2N; UBE2N -3.76 Apurinic endonuclease; APE; APE1; APEX -2.59 MMC + BVDU recovery, with BVDU vs. MMC r. without BVDU MMC recovery without BVDU (=1) Platelet-activating factor acetylhydrolase, isoform 1B, subunit; PAFAH1B1 +2.83 U5 snRNP-specific protein, 116-KD +2.62 Hemoglobin-ß locus; HBB +5.70 Hemoglobin- locus 1; HBA1 +5.85 Actin, ß; ACTB +2.77 Similar to ß-actin +3.72 Actin-like +3.55 Tropomodulin 2; TMOD2 +2.95 Succinate dehydrogenase complex, subunit A, Flavoprotein; SDHA absent vs. 0.26 Pyruvate dehydrogenase complex, E1- polypeptide 1; PDHA1 -3.29 Tubulin, ß-5 -3.03 Poly(rC)-binding protein 2; PCBP2 -3.90 Malic enzyme 2; ME2 -3.02 Mini chromosome maintenance deficient 7; mitotin, cell division cycle-like 1; CDCL1 -3.14