Figure 1: Effects of cisplatin on survival of lung cancer cells.

Transcription

1 Figure 1 Figure 1: Effects of cisplatin on survival of lung cancer cells. To determine the IC 50 concentration of cisplatin, cells were treated with various concentrations of cisplatin and cell survival was assessed by colony forming assay.

2 Table 2: List of genes analyzed by real time RT-PCR. Anti-apoptotic genes Pro-apoptotic genes ABL1, AKT1, BAG1, BAG3, BCL2, BCL2A1, BCL2L1, BCL2L10, BCL2L2, BFAR, NAIP, BIRC2, BIRC3, BIRC4 (XIAP), BIRC6, BIRC8, BNIP1, BNIP2, BNIP3, BRAF, CASP2, CFLAR, FAS (TNFRSF6), IGF1R, MCL1, TNF, CD27 APAF1, BAD, BAG4, BAK1, BAX, BCL10, BCL2L11, BCLAF1, BID, BIK, BNIP3L, NOD1, CARD6, CARD8, CASP1, CASP10, CASP14, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CD40, CD40LG, CIDEA, CIDEB, CRADD, DAPK1, DFFA, FADD, FASLG, GADD45A, HRK, LTA, LTBR, NOL3, PYCARD, RIPK2, TNFRSF10A, TNFRSF10B, TNFRSF11B, TNFRSF1A, TNFRSF21, TNFRSF25, TNFRSF9, TNFSF10, CD70, TNFSF8, TP53, TP53BP2, TP73, TRADD, TRAF2, TRAF3, TRAF4

3 Table 3. Statistical analysis for Figure 1: The following hypothesis was tested for repeatedly measured surviving fraction as an outcome of measure for each of the cell lines. Hypothesis: There is no difference in slope of surviving fraction of cells between high and low doses. Cell*** Dose* H-157 High vs. Low UKY-29 High vs. Low A459 High vs. Low H460 High vs. Low Threshold (dose Gy) Estimate** Standard Error** P-value** * Note that dose levels as low and high are consistent among different cell lines. For threshold value of 0.5Gy: low dose corresponding to any dose <0.5Gy and high dose corresponding to any dose >0.5Gy. For threshold value of 1Gy: low dose corresponding to any dose <1Gy and high dose corresponding to any dose >1. ** Estimate, its standard error and p-value for a given cell and threshold were calculated from fitting a repeated measures analysis of variance to surviving fractions. Repeatedly measured surviving fractions at each dose level as well as over different doses were taking into account by including autoregressive variance-covariance matrix in the models. Type-I error was set to 5%. ***Overall, there were statistically significant differences in slope of surviving fraction between high and low doses for cell lines H-157, UKY-29, A459. However, there were no statistically significant differences in slope of surviving fraction between high and low doses for cell line H460.

4 Table 4. Statistical analysis for Figure 2: The following hypothesis was tested for surviving fraction as an outcome of measure for two cell lines at a given specific dose level. Hypothesis: There are no differences in means surviving fractions among different fractionations at a given cell line. Cell*** Dose (Gy)* Fractionation Standard (dose per Estimate** Error** fraction) P-value** H Gy Gy Gy Gy vs. 1Gy Gy vs. 2Gy Gy vs. 2Gy Gy Gy Gy Gy vs. 1Gy Gy vs. 2Gy Gy vs. 2Gy Gy Gy Gy Gy vs. 1Gy G 0.5Gy vs. 2Gy Gy vs. 2Gy UKY Gy Gy Gy Gyvs 0.5Gy vs. 1Gy Gy vs. 2Gy Gy vs. 2Gy Gy Gy Gy Gy vs. 1Gy Gy vs. 2Gy Gy vs. 2Gy Gy Gy Gy Gy vs. 1Gy Gy vs. 2Gy Gy vs. 2Gy

5 * Note that only 0Gy, 2Gy, 4Gy, 6Gy dose levels are common to all different fractionation groups i.e. 0.5Gy, 1Gy and 2Gy dose per fraction. Therefore, the analysis was restricted only to 0Gy, 2Gy, 4Gy, 6Gy dose levels. Since there is no variation in surviving fraction at dose=0gy (i.e. SF=1 for all fractionation schemes) no test can be done. ** Estimate, its standard error and p-value for a given cell and the dose level were calculated from fitting one-way analysis of variance (one-way ANOVA) to surviving fractions with different fractionation schemes Type-I error was set to 5%. The p-values for pairwise group comparisons i.e. 0.5Gy vs. 1Gy dose per fraction, 0.5Gy vs. 2Gy dose per fraction and 1Gy vs. 2Gy dose per fraction should be compared to 1.66% due to Bonferroni correction to multiple comparison. Bold p-values are the one that are NOT statistically significant. ***Overall, all the fractionation schemes were statistically significant and 2Gy dose per fraction scheme had the higher surviving fraction in both cells for all doses but not for UKY-29 at a total dose of 4Gy and 6Gy. Notice that the mean estimate for UKY-29 at a total dose of 4Gy for 2Gy dose per fraction and at dose=0.5gy were very close to each other. There were statistically significant differences between different fractionation schemes when we performed pairwise comparisons such as 0.5Gy vs. 1Gy dose per fraction, 0.5Gy vs. 2Gy dose per fraction and 1Gy vs. 2Gy dose per fraction at a given dose level for each of the cell lines. The bold p-values listed in the table are the ones that are NOT statistically significant i.e. there is no significant differences between the mean surviving fraction for given two groups for that particular cell line and the dose level.

6 Table 5: Chemo-enhancement ratios of LDFRT and 2Gy in H-157 and UKY-29 cells following incubation with cisplatin either immediately before (IB) or 24h before (24hB) irradiation obtained from colony forming assays. Cell line 2Gy CER LDFRT IB 24hB IB 24hB UKY H

7 Table 6: Up- and down-regulated genes compared to untreated group (more than 50 fold differences) in lung cancer cells by real time RT PCR analysis. Treatment H-157 UKY-29 H460 A549 Up Down Up Down Up Down Up Down Cisplatin NAIP CASP5 CD40LG CIDEA CIDEB (p) FASLG NOL3 TNFSF10 2Gy NAIP CD40LG CIDEA CIDEB FASLG NOL3 TNFSF10 Cisplatin+2Gy NAIP CASP5 CD40LG CIDEA CIDEB FASLG NOL3 LDFRT FASLG (p) - - NAIP CASP5 CD40LG CIDEA CIDEB FASLG NOL3 Cisplatin+LDFRT BAG3 NAIP CASP5 CD40LG CIDEA FASLG NOL3 TRAF4 (a) TNFRSF11B (p) TNFRSF11B NOD1 (p) TNFRSF11B

8 Table 7: List of the modulated genes corresponding to Figure 5: Up- and down- regulated anti and pro- apoptotic genes in H-157 (A), UKY-29 (B) and A549 (C) cells compared to H460 cells by real time RT PCR analysis.. A: H-157 vs. H460 Anti-apoptotic genes Pro-apoptotic genes Treatment Downregulated Upregulated Downregulated Upregulated Cisplatin BCL2L10, BIRC2, BIRC8, CD27 NAIP LDFRT BIRC3 BAG3, BCL2L1, BIRC8, CASP2, NAIP 2Gy Cisplatin+LDFRT Cisplatin+2Gy BCL2A1, BCL2L10, BIRC2, BIRC3, BIRC6, BIRC8, BRAF, CD27, FAS, NAIP, TNF BCL2L10, BIRC8, BRAF, FAS, TNF BCL2L1, BCL2L2, CASP2 CARD6, CIDEB, LTA, TNFSF8, TP73 LTA CARD6, CIDEB, DAPK1, FASLG, HRK, LTA, NOD1, PYCARD, TNFRSF25, TNFSF8, TP73 CARD6, CASP10, CASP14, CIDEB, DAPK1, LTA, PYCARD, TNFSF8 BCL10, CARD8, CASP1, CASP10, CASP6, CASP8, CD40, DAPK1, FASLG, NOD1, TNFRSF9, TNFSF10 BAD, BCL2L11, CASP14, CASP5, CASP6, CD4, CD40LG, CIDEB, DAPK1, FADD, FASLG, NOD1, NOL3, TNFRSF25, TNFSF10, TP53, TP73,TRAF2 CASP5, CASP6, TNFSF10 TNFSF10 BAD, BIK, CASP5, CASP6, CD40, LTBR, NOD1, NOL3, TNFRSF9, TNFSF10, TRAF2

9 B: UKY-29 vs. H460 Anti-apoptotic genes Pro-apoptotic genes Treatment Down- Upregulated Down- Upregulated regulated regulated Cisplatin AKT1, BAG3, BCL2, BCLAF1, BCL2A1, BCL2L1, CD40 BCL2L10, BCL2L2, BIRC2, BIRC3, BIRC6, BIRC8, BNIP1, BRAF, CASP2, CD27, CFLAR, LDFRT IGF1R, MCL1, NAIP, TNF BAG3, BCL2A1, BCL2L10, BIRC3, CD27, NAIP, TNF APAF1, BAD, BAG4, BAK1, BAX, BCL10, BCL2L11, BID, CARD8, CASP10, CASP14, CASP5, CASP6, CASP7, CASP9, CD40LG, CIDEB, CRADD, FADD,F ASLG, GADD45A, HRK, LTA, LTBR, NOD1, NOL3, PYCARD, RIPK2, TNFRSF10A, TNFRSF1A, TNFRSF21, TNFRSF25, TNFRSF9, TNFSF10, TNFSF8, TP53, TP53BP2, TP73, TRADD, TRAF2, TRAF3 BCL2L11, CASP5, CD40LG, CIDEB, FASLG, LTA, NOL3, PYCARD, TNFRSF1A, TNFRSF25, TNFSF10, TNFSF8, TP73, TRAF3 2Gy AKT1, BAG3, BCL2, APAF1, BAD, BAK1, BAX, BCL10, BCL2A1, BCL2L1, BCL2L10, BCL2L2, BIRC2, BIRC3, BIRC6, BIRC8, BNIP1, BRAF, CASP2, CD27, CFLAR, IGF1R, MCL1, NAIP, TNF Cisplatin+LDFRT BAG3, BCL2A1, BCL2L, BCL2L10, BCL2L2, BIRC3, CD27, NAIP, TNF BCL2L11, BID, CARD6, CARD8, CASP1, CASP10, CASP14, CASP5, CASP6, CASP9, CD40, CD40LG, CIDEB, CRADD, FADD, FASLG, GADD45A, HRK, LTA, LTBR, NOD1, NOL3, PYCARD, RIPK2, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF21, TNFRSF25, TNFRSF9, TNFSF10, TNFSF8, TP53, TP53BP2, TP73, TRADD, TRAF2, TRAF3, TRAF4 BAX, BCL2L11, CASP5, CD40LG, CIDEB, FASLG, LTA, NOD1, NOL3, PYCARD, TNFRSF10A, TNFRSF1A, TNFRSF25, TNFRSF9, TNFSF10, TNFSF8, TP73, TRAF2, TRAF3, TRAF4 Cisplatin+2Gy AKT1, BAG3, BCL2, BIK,CASP6, APAF1, BAD, BAG4, BAK1, BAX, BCL10, BCL2A1, BCL2L1, BCL2L10, BCL2L2, BIRC3, BIRC6, BNIP1, BRAF, CASP2, CD27, CFLAR, FAS, IGF1R, MCL1, NAIP, TNF CASP8 BCL2L11, BID, CASP14, CASP5, CASP7, CD40LG, CIDEB, CRADD, FADD, FASLG, GADD45A, HRK, LTA, NOD1, NOL3, PYCARD, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF25, TNFRSF9, TNFSF10, TNFSF8, TP53, TP53BP2, TP73, TRADD, TRAF2, TRAF3

10 C: A549 vs. H460 Anti-apoptotic genes Pro-apoptotic genes Treatment Downregulated Upregulated Downregulated Upregulated Cisplatin BCL2A1, NAIP BCLAF1, CIDEB, LTA, ABL1, BAK1, BCL10, BCL2L11, BCL2L10, BIRC3, TNFRSF11, TNFSF8, BID, BIK, CASP10, CASP14, BIRC4, BIRC8, TRAF3 CASP6, CASP8, CD40, CD70, BNIP2, BRAF, FAS DAPK1, FADD, GADD45A, NOL3, PYCARD, TNFRSF10A, TNFRSF10B, TNFRSF9, TNFSF10, TP53, TRAF2, TRAF4 LDFRT LTA NOD1 2Gy BAG3, BCL2A1, CASP5, CD40LG, TNFSF10 BIRC3, BIRC6, CIDEB, FASLG, LTA, BIRC8, BRAF, NOD1, TNFRSF11B, CD27, FAS, NAIP TNFSF8 Cisplatin+LDFRT BIRC8 LTA, TNFRSF11B CASP14 Cisplatin+2Gy BCL2L10, BIRC3, BCL2L1 BCLAF1, CASP10, BIK, CASP5, CASP6, CD40, BIRC4, BNIP2, CD40LG, NOL3, TNFRSF9, BRAF, FAS TNFSF10, TP73 CIDEB, DAPK1, FASLG, LTA, TNFRSF11B, TNFSF8