1. Quest for the missing gene: Tumor suppressor on 3p21? Tatiana Dracheva LPG

2. Introduction 3p21 region is often missing in Lung Cancer Our previous data (LOH) show a significant correlation between the loss of the specific part of 3p21 (D3S1581 - D3S1289) and the early onset of cancer. We limited the region of loss to ~2Mb (48.5- 50.5 Mb) Possible telomerase suppressor gene is located in this region.

3. Telomerase and Cancer Telomeres are the repeated sequences located on both ends of chromosomes in eukaryotes In normal cells telomeres are shorten with each cell division Tumor cells show activation of telomerase, a ribonucleoprotein enzyme that maintains telomere length by addition of the sequences of TTAGGG repeats to telomeres. Tanaka et al suggested the possible telomerase supressor gene located on 3p21 (Tanaka H, Shimizu M, Horikawa I, Kugoh H, Yokota J, Barrett JC, Oshimura M. Evidence for a putative telomerase repressor gene in the 3p14.2-p21.1 region. Genes Chromosomes Cancer. 1998 )

4. Map of LOH on 3p

5. LOH at Chr. 3p markers with age of lung cancer onset (20pairs 79)

6. LOH at 3p21 with age of lung cancer onset (19pairs 79)

7. Goals of the study Narrow down the LOH region on 3p21 Find candidates for the tumor supressor gene on 3p21 Find candidates for the telomerase supressor gene

8. TaqMan® Low Density Array - micro fluidic card Uses TaqMan® Gene Expression Products, pre- designed TaqMan probe and primer sets for human, mouse, and rat gene expression research using real-time PCR Provides convenient access to 384-assay format without liquid handling robotics Screens multiple samples against custom-selected gene panels Minimizes reagent consumption

9. Schematic representation of the TaqMan principle A: Primers and probe anneal to the target gene. Fluorescence emission does not occur because the probe is still intact. B: During the extension phase of the PCR reaction, the probe is cleaved by the 5'–3' exonuclease activity of the Taq polymerase, allowing fluorescence emission. FW, forward; RV, reverse; F, fluorophore; Q, quencher dye.

10. PCR amplification plot Fluorescence emission is measured continuously during the PCR reaction and Rn (increase in fluorescence emission, from which the background fluorescence signal is subtracted) is plotted against cycle number. The threshold cycle (Ct) is the cycle at which the fluorescence exceeds a chosen threshold.

11. Samples/Arrays Experiment “LOH”: 39 tumor-normal pairs used for the LOH analysis After transcription and quality control : 17 pairs + 9tumors +1normal +4 repeats = 48 arrays we put on the cards. Among 17 pairs: 7 pairs belong to the “young” group, 10 - to the “old” Experiment “Telomerase repressor”: 3 samples or RCC23 cell line: 1 parental (active telomerase), 2 with transferred normal human chromosome 3 (repressed teromerase activity) Card layout: 68 probes from 3p21, 26 control probes from the “162 list”, 18S control, telomerase reverse transcriptase (TERT)

12. TaqMan ® Low Density Array Format

13. Analysis Normalization: 18S Ct extraction or Average of 26 probes from “162 list” extraction. Paired T-test Normal vs. Tumor to confirm up- or down- regulation of 26 probes Cluster -type representation in order to see LOH region T-test Tumor Old vs Young Search for genes absent in parental RCC23 but present in modified RCC23+3

14. Paired T-test Normal vs. Tumor to confirm up- or down-regulation of 26 probes

15. Cluster -type representation of all samples

16. Cluster -type representation of Tumor-Normal (paired only)

17. Cluster -type representation in order to see LOH region (Tumors only)

18. T-test Tumor Old vs Young

19. Candidates for the telomerase supressor

20. Results/Future work We were not able to further limit the LOH region with the Q-PCR method. ( Tumor samples contain too much normal. Small sample set.) We found candidates genes for the telomerase supressor on 3p21. We are sequencing candidate genes looking for the mutations. Our collaborators are introducing those genes to the RCC23 in order to see if one of the indeed supress telomerase.

21. Aknolegments Jin Jen Felicia Mann Daoud Meerzaman Izumi Horikawa Carl Barrett Steve Hoffmann (NIDDK)

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