The SP013 gene, required for meiosis I segregation in Saccharomyces cerevisiae, produces two developmentally regulated transcripts (1.0 and 1.4 kilobases) that differ in length at their 5' ends. The shorter transcript is sufficient to complement the spol3-1 mutation and contains a major open reading frame encoding a highly basic protein of 33.4 kilodaltons. A fragment upstream (-170 to -8) of the open reading frame confers meiosis-specific transcription on a spol3-HIS3 fusion. Deletions at the 5' end of spo3-lacZ fusions define a region between -140 and -80 that is essential for meiosis-specific expression. This region acts in an orientation-independent manner and is responsive to the MAT-RME regulatory cascade. It contains a 10-base-pair sequence, TAGCCGCCGA, found in a number of meiosis-specific genes, that appears to be required for SP013 expression. This sequence is identical to URS1, a ubiquitous mitotic repressor element.Initiation of meiosis and spore formation in Saccharomyces cerevisiae is controlled by cell type and nutritional status (1, 2). Cell-type control occurs through the products of the MATa and MATa loci, which combine to negatively regulate transcription of RMEI (3, 4). RMEI negatively regulates IMEI, whose expression appears to be sufficient to induce meiosis (5). IMEI is also under negative control by the system monitoring nutritional status (5, 6).SP013 is required for the proper separation of homologs at meiosis I (7). SP013 has been cloned and encodes two overlapping transcripts with the same 3' end (8 MATERIALS AND METHODS Strains. RE944 (spol3-1/spol3-1 ura3/ura3) is from our strain collection. LP112 (MATa/MATa ade2/ade2 canl-100/ canl-100 his3-1 1, 15/his3-1 1,15 leu2-3,1 12/leu2-3, 112 trpl-1/ trpl-l ura3-1/ura3-1), obtained from S. Lindquist (University of Chicago), is a cross of W303-1A and W303-1B from R. Rothstein (Columbia University). Isogenic diploids SFY67 (MATa/MATa) and SFY68 (MATa/MATa) were obtained from J. Segall (University of Toronto). S104 (MATa/MATa leul/LEUI ura3/ura3 trpl/trpl cani/cani his4-519/HIS4 RMEI/rmel::LEU2), S150 (MATa/MATa leu2/leu2 ura3/ ura3 trpl/trpl his4-519/his4-712 canl/CANl lysliL YSI RMEl/rmel::LEU2), S151 (MATa/MATa leu2/leu2 ura3/ ura3 trpl/trpl his4-519/his4-712 canl/CANI lysl/L YSI rmel ::LEU2/rmel ::LEU2), S152 (matal-50/MATa leu2/ leu2 ura3/ura3 trpl/trpl his4-519/his4-712 canl/CANI lysl/ LYSI RMEJ/rmel ::LEU2), and S153 (matal-50/MATa leu2/leu2 ura3/ura3 trpl/trpl his4-519/his4-712 canl/CANI lysl/LYSI rmel::LEU2/rmel::LEU2) were acquired from A. Mitchell (Columbia University).Growth and Sporulation. General methods for growth and sporulation have been described (7,8,14). Strains were grown to midlogarithmic phase in synthetic acetate medium [1% potassium acetate plus 0.68% Difco yeast nitrogen base without amino acids, buffered to pH 6.5 with 0.05 M potassium phthalate (pH 5.5) and supplemented with auxotrophic requirements] prior to sporulation.DNA Sequence Analysis. Dideoxy sequencing of the cloned SP013 gene was carried out as ...

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The primary objective of this study is to identify prognostic site-specific epigenetic changes in surgically treated Stage I and II nonsmall cell lung cancer (NSCLC) patients by quantifying methylation levels at multiple CpG sites within each gene promoter. Paraffin-embedded tumors from stage Ib, IIa and IIb in training and validation groups of 75 and 57 surgically treated NSCLC patients, respectively, were analyzed for p16, MGMT, RASSF1, RASSF5, CDH1, LET7, DAPK and PTEN promoter hypermethylation. Hypermethylation status was quantified individually at multiple CpG sites within each promoter by pyrosequencing. Molecular and clinical characteristics with time to recurrence (TTR) and overall survival (OS) were evaluated. Overall average promoter methylation levels of MGMT and RASSF1 were significantly higher in smokers than in nonsmokers (p 5 0.006 and p 5 0.029, respectively). Methylation levels of the p16 promoter were significantly higher in squamous cell carcinoma than in adenocarcinoma (p 5 0.020). In univariate analysis, hypermethylation of RASSF1 at CpG sites 253 and 248 and PTEN at CpG site 21310 were the significantly associated with shorter TTR (p 5 0.002 and p < 0.000, respectively). Hypermethylation of PTEN at 21310 and DAPK at 21482 were most significantly associated with outcome in multivariate analysis. These results show that methylation of specific promoter CpG sites in PTEN, RASSF1 and DAPK is associated with outcome in early stage surgically treated NSCLC.

Identification of objective tumor regressions with epidermal growth factor receptor tyrosine kinases (EGFR TKI) in non^small cell lung cancer (NSCLC) patients has resulted in intense, worldwide clinical and basic research directed toward finding the optimal use of EGFR TKIs in NSCLC. EGFR TKI clinical trials have shown that higher response rates and longer survival are associated with specific patient characteristics and that using conventional chemotherapy simultaneously with EGFR TKIs in unselected patients does not increase survival. Molecular studies have revealed that EGFR-activating mutations and high EGFR gene copy number are frequently found in patients who have the best outcomes with EGFR TKIs. More recent studies suggest that KRAS mutations may identify the subset of patients who have the worst outcome with the EGFR TKI treatment. Currently, investigators are trying to determine the optimal approach to selecting patients for treatment with EGFR TKIs. Studies that have evaluated the potential predictive value of clinical features and/or molecular profiles in EGFR TKI-treated NSCLC patients are discussed in this review.Preclinical studies with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKI) suggested that these agents would be cytostatic (1, 2). The observation of significant, often dramatic, tumor regressions induced by EGFR TKIs in heavily pretreated patients with advanced non -small cell lung cancer (NSCLC) was therefore unexpected (3, 4). Since those initial observations, there have been several revealing findings with EGFR TKIs, including discovery of higher response rates in never smokers (5), Asians (6), women (6 -9), and patients with adenocarcinomas (7 -9). Similarly, the negative results in phase III trials comparing chemotherapy doublets plus an EGFR TKI versus chemotherapy alone (10 -13) were particularly disappointing because preclinical observations (14) and theoretical considerations suggested a relatively high chance of success with this strategy.The observation of higher response rates with EGFR TKIs in selected groups of patients, as well as the disappointing results with simultaneous chemotherapy and EGFR TKIs in unselected patients, led lung cancer researchers to study the potential predictive value of molecular profiles in patients treated with EGFR TKIs. However, negative findings about the predictive value of EGFR protein expression in gefitinib-treated patients raised considerable doubt about the role of molecular profiles in patient selection (15). With the discovery of EGFR-activating mutations in tumors from most patients who had EGFR TKIinduced tumor responses (16,17), skepticism was soon replaced by enthusiasm for molecular profile research in patients treated with EGFR TKIs. There is increasing evidence that EGFR mutations (18 -21) and high EGFR gene copy number (22, 23) are associated with higher response rates and longer survival.Although clinicians are delighted to have an effective new treatment and new insights about lung cancer biology, ...

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Some well-known fatty acid ester surfactants, e.g., Cremophor EL and Solutol HS 15, are modulators of multidrug resistance in vitro and in vivo. Because they are polydisperse, and their active component(s) have not been identified, the therapeutic potential of such surfactants is unclear. To better define the active components of Solutol HS 15 and to make more potent surfactant multidrug resistance modulators, highly purified C-18 fatty acids were esterified with ethylene oxide at 5-200 molar ratios. Unexpectedly, ethylene oxide esters of pure 12-hydroxy stearic acid, the major components of Solutol HS 15, displayed negligible resistance modification activity compared with Solutol HS 15 itself or to stearic and oleic acid esters synthesized under identical conditions. Since oleic acid esters appeared to have good activity, a series of these compounds was prepared to determine the optimal ethylene oxide/fatty acid ratio. The optimal ratio was found to be 20 mole ethylene oxide: I mole fatty acid, with a steep decline in activity for products made with ratios above and below the optimum. The most active oleic acid ester, designated CRL 1337, was 8.4-fold as potent as Solutol HS 15 and over 19-fold as potent as Cremophor EL in promoting rhodamine 123 accumulation in multidrug-resistant KB 8-5-11 cells in vitro. Our results show that the structure of the hydrophobic domain (fatty acid) of surfactants as well as its hydrophile-lipophile balance are critical in determining the potency of surfactants as reversing agents.

Fatty acid ester surfactants Cremophor EL and Solutol H S I 5were described earlier as modulators of multidrug resistance mediated by MDR I P-glycoprotein (Pgp). We have shown that the most active components of these polydisperse surfactants are fatty acid-polyethylene glycol-fatty acid diesters (FA-PEG-FA). A new generation of Pgp-surfactant inhibitors of defined structure was therefore synthesized. In the present study we show that these compounds are also able to inhibit upregulation of MDRI gene expression caused by cytarabine (AM-C) and doxorubicin in human tumor cell lines H9 and KB 3-I, which express minimal levels of MDR I mRNA. The surfactant inhibitors, however, had no effect on the induction of Q 1996 Wiley-Liss, Inc.

Purpose: Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, potentiates antitumor effects of erlotinib in preclinical studies, and COX-2 is frequently expressed in non^small cell lung cancer (NSCLC).With these observations, we designed a phase II trial to evaluate the efficacy and safety of erlotinib plus celecoxib in advanced NSCLC. Experimental Design: Previously treated stage IIIB/IV NSCLC patients were given celecoxib at 400 mg orally twice daily and erlotinib at 150 mg orally daily until disease progression. Planned accrual was 40 patients. Tissue was collected for epidermal growth factor receptor (EGFR) analysis and COX-2 immunohistochemistry. Results: Twenty-six patients were enrolled (17 men, 9 women; median age, 66 years). Eighteen and 21patients had tissue available for EGFR analysis and COX-2 immunohistochemistry, respectively. The median progression-free survival (PFS) and overall survival were 2.0 and 9.2 months, respectively. Eleven of 21 patients tested had increased tumor COX-2 expression, which was strongly associated with prolonged PFS (P = 0.048). Four patients on anticoagulation or with a history of peptic ulcer disease had grade 3/grade 4 upper gastrointestinal bleeding (GIB), prompting early study closure. Three patients with GIB had endoscopy that found peptic ulcers. Conclusions: The combination of erlotinib and celecoxib does not seem superior to erlotinib alone in unselected patients. However, longer PFS with high-tumor COX-2 expression suggests that trials of EGFR and COX-2 inhibitors may be warranted in this patient subset. GIB observed in our trial supports excluding patients with a history of peptic ulcer disease or those requiring therapeutic anticoagulation from future EGFR and COX-2 inhibitor studies.