Expression Profiling Identifies Three Pathways Altered in ... - CiteSeerX

Journal of Gerontology: BIOLOGICAL SCIENCES 2006, Vol. 61A, No. 9, 879–889

Copyright 2006 by The Gerontological Society of America

Expression Profiling Identifies Three Pathways Altered in Cellular Immortalization: Interferon, Cell Cycle, and Cytoskeleton Aviva Levine Fridman,1 Lin Tang,1 Olga I. Kulaeva,1 Bin Ye,1 Qunfang Li,1 Fatimah Nahhas,1 Paul C. Roberts,2 Susan J. Land,1 Judith Abrams,3 and Michael A. Tainsky1 1

Abrogation of cellular senescence, resulting in immortalization, is a necessary step in the tumorigenic transformation of a cell. Four independent, spontaneously immortalized Li-Fraumeni syndrome (LFS) cell lines were used to analyze the gene expression changes that may have given these cell lines the growth advantage required to become immortal. A cellular senescence–like phenotype can be induced in immortal LFS cells by treating them with the DNA methyltransferase (DNMT) inhibitor 5-aza-deoxycytidine. We hypothesized, therefore, that genes epigenetically silenced by promoter methylation are potentially key regulators of senescence. We used microarrays to compare the epigenetic gene expression profiles of precrisis LFS cells with immortal LFS cells. Gene ontology analysis of the expression data revealed a statistically significant contribution of interferon pathway, cell cycle, and cytoskeletal genes in the process of immortalization. The identification of the genes and pathways regulating immortalization will lead to a better understanding of cellular immortalization and molecular targets in cancer and aging.

N

ORMAL human diploid cells divide a finite number of times before senescing. Replicative senescence, originally described by Hayflick (1), is a state of arrested growth and altered function that occurs in normal somatic cells after a finite number of cell divisions in culture (2,3). Senescent cells are arrested in G1 phase of the cell cycle, with a large, flattened morphology. Although senescent cells are metabolically active (4,5), they are unable to synthesize DNA, even in response to stimulation with growth factors. In somatic cell hybrids the senescent phenotype is dominant over the immortal phenotype (4,6), indicating that losses in gene expression may be the more critical events in cellular immortalization. Four senescence complementation groups were identified, suggesting that there are at least four senescence genes or gene pathways (4,6). Cancer cells have the ability to grow indefinitely because they have bypassed replicative senescence and become immortal. There are a variety of cellular factors and processes involved in the immortalization of a cell, including telomere length stabilization, genomic instability, and epigenetic gene silencing by selective promoter methylation (7). Carcinogenesis is a multistep process arising from the accumulation of mutations in tumor suppressor genes and oncogenes that confer growth advantages and/or genomic instability to the cell, which leads to further mutations. Understanding the mechanisms by which cells bypass senescence and become immortal could greatly improve the treatment and/or prevention of cancer, particularly at the earliest stages.

In 75% of Li-Fraumeni syndrome (LFS) families, the inheritance of heterozygous mutations in the p53 tumor suppressor gene results in a significantly increased likelihood of developing multiple types of cancer. Cell lines derived from patients with LFS divide like normal fibroblasts for approximately 30 population doublings. These cells we define as precrisis cells. As a consequence of the germline mutations in p53, precrisis LFS cells develop genomic instability and immortalize, thereby providing a model for spontaneous immortalization that can be used to study escape from senescence (8). The advantage of these cell lines over other immortalization cell systems is that immortalization occurs spontaneously, and therefore is not due to the addition of chemicals which might cause irrelevant mutations or by viral oncogenes which alter gene expression patterns beyond those relevant to immortalization. We used these cell lines to characterize the gene(s) and pathway(s) involved in senescence and immortalization and to profile the contribution of gene silencing, via DNA methylation, to these processes. Although human cells from normal donors rarely, if ever, spontaneously immortalize in culture, these LFS cell cultures do so without intervention (8–10), and do so more rapidly when treated with physical or chemical agents (11,12). Recently, fibroblasts derived from colonic stroma of a familial adenomatous polyposis patient were also shown to spontaneously immortalize in culture (13). Loss of the wild-type p53 allele and stabilization of telomere length in LFS human fibroblasts are essential steps, albeit not sufficient, for spontaneous cellular immortalization. The 879

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Program in Molecular Biology and Human Genetics, Barbara Ann Karmanos Cancer Institute, and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan. 2 Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan. 3 Biostatistics Core, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan.

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LFS cell lines than expected statistically. We also identified 14 genes that were epigenetically repressed during immortalization in all four immortal LFS cells lines. MATERIALS AND METHODS

Cell Culture, Genotyping, and Viral Sensitivity Assay The cell lines MDAH041 (p53 frameshift mutation) and MDAH087 ( p53 missense point mutation) were derived from primary fibroblasts by skin biopsy from a female and male patient, respectively, with LFS (8). The Tainsky Lab developed four independent, spontaneously immortalized LFS cell lines: one immortal cell line from MDAH041 and three independent immortal cell lines derived from MDAH087 (MDAH087-1, MDAH087-10, and MDAH087N) (10). All the cells were cultured at 378C in 5% humidified CO2 in modified Dulbecco’s Eagle’s medium (Gibco BRL, Gaithersburg, MD) with 10% fetal bovine serum and penicillin at 500 U/mL, streptomycin at 100 lg/mL. The appropriate regions in the p53 gene containing the mutation were sequenced in precrisis and immortal cell lines to confirm heterozygosity in the precrisis cell lines and loss of the remaining wild-type p53 mutation in the immortal cell lines. The viral sensitivity assay was performed as described (27). Results from using Prism software (GraphPad, Inc., San Diego, CA) are expressed as percent cell cytolysis compared to control uninfected cells. 5-aza-dC and IFN-a Treatment, RNA Isolation, and the Affymetrix Microarray Assays Precrisis (population doubling [PD] 11–22) and immortal (PD . 200) MDAH041 and MDAH087 fibroblasts were treated with 5-aza-dC as described (18). After Day 6 the cells were cultured in drug-free medium. On Day 8 total RNA was extracted using the QIAGEN RNeasy Kit (QIAGEN, Inc., Valencia, CA). Preparation of cDNA and Affymetrix (Santa Clara, CA) oligonucleotide microarray processing was performed by the Wayne State University and Michigan Center for Genomic Technologies Applied Genomics Technology Center in accordance with Affymetrix protocols. Cells were cultured in media supplemented with IFN-a at 1000 U/mL for 8 or 30 hours. Following treatment, total RNA was isolated for real-time reverse transcription– polymerase chain reaction (Q-RT-PCR) as described below. For experiments of IFN-a and/or 5-aza-dC combined treatments, cell cultures were treated under the following conditions: Cells were cultured in media either containing 1 lM 5-aza-dC or IFN-a at 1000 U/mL for 2 days, and on Day 3 cell media was replaced with drug-free media. A third set of cells was cultured in media containing 1 lM 5-aza-dC for 2 days, and on Day 3 media was replaced with media supplemented with IFN-a at 1000 U/mL. On Day 5, total RNA was isolated for Q-RT-PCR as described below. Analysis of Microarray Data Microarray experiments on MDAH087 were performed using the Affymetrix HGU95Av2 GeneChip containing 12,625 probes. Three RNA preparations from MDAH087-N,


absence of p53 does, however, extend the life span of these cells (8,12,14,15). Immortal cells maintain stable telomere lengths by either activating telomerase or by alternative lengthening of telomeres (ALT) (16,17). Because the loss of the wild-type p53 allele and the stabilization of telomere length are insufficient to cause immortalization (15), we have sought to understand the additional genetic events required for immortalization to occur. In a preliminary study using one LFS cell line, MDAH041, we determined that among the genes epigenetically silenced during immortalization, a statistically significant portion of them were associated with the interferon (IFN) signaling pathway (18). In this study we used four immortal LFS cell lines, MDAH041, and three independently immortalized LFS cells lines derived from the fibroblasts of a second LFS patient, MDAH087, to identify genes, pathways, and chromosomal regions involved in immortalization that are common to all of the cell lines studied. Methylation of DNA is an epigenetic modification that occurs almost exclusively at CpG sites. It is involved in a variety of normal cellular processes, including developmental imprinting, X-chromosome inactivation, and tissuespecific gene expression, as well as tumorigenesis (19–22). Although CpG sites are typically methylated, CpG-rich regions of DNA, about 200 to 2000 bp in length, referred to as CpG islands) are generally unmethylated. Abnormal DNA cytosine methylation of a CpG island(s) in the promoter of a tumor suppressor gene and the subsequent silencing of the gene’s expression are common epigenetic changes that occur in human cancers (22). Genes shown to be hypermethylated in cancer are found in diverse pathways, including DNA repair, cell-cycle control, invasion, and metastasis. There are a number of well established tumor suppressor genes that are hypermethylated in cancer, including p16INK4a, p15INK4b, p14ARF, p73, APC, and BRCA1 (19). The frequency of hypermethylation of genes is tumor type-specific (19) and can be reversed by the demethylation of DNA using chemical agents. Our previous study was the first to demonstrate pathway-specific epigenetic changes as a preneoplastic event (18). An inhibitor of DNA methyltransferase (DNMT), 5aza-deoxycytidine (5-aza-dC), is a cytosine analog that incorporates at cytosine residues in DNA during replication. DNMTs are trapped by covalently binding with 5-aza-dC, resulting in the depletion of DNMTs after several rounds of replication. The depletion of DNMTs leads to DNA hypomethylation and the reactivation of genes previously silenced by methylation (23–25). The expression of genes aberrantly silenced in immortal cells can be restored by treatment with 5-aza-dC (18,26). In this study we used the four independent, spontaneously immortalized cell lines, derived from two LFS patients, MDAH041 and MDAH087, to identify genes and pathways involved in immortalization, expanding our studies beyond those pathways that are unique to a single cell line. MDAH041 and each of the three immortal MDAH087 cell lines had significantly more IFN-regulated genes with changes in expression than expected by chance. Additionally, significantly more cell cycle and cytoskeletal genes were regulated during immortalization in all four immortal

PATHWAYS OF CELLULAR IMMORTALIZATION

Identification of CpG Islands in Genes To detect CpG islands in the promoter region of genes, we identified 1000 bp to þ500 bp of the transcription start site using UCSC Golden Path (http://genome.ucsc.edu). We then used MethPrimer software (http://mail.ucsf.edu/ ;urolab/methprimer/index1.html) to identify CpG islands. MethPrimer default values for observed ratio of C plus G to CpG, and minimum average percentage G plus C were used, .0.6 and .50, respectively. Confirmation of Gene Expression Changes by Q-RT-PCR The primary fibroblasts cell lines CRL-1502 (American Type Culture Collection [ATCC] CRL-1502; Manassas, VA) and CCL-110 (ATCC CCL-110) were obtained by skin biopsy from normal donors. The senescing HCA2 cells were a gift from Dr. James R. Smith (University of Texas Health Science Center at San Antonio, Barshop Center for Longevity and Aging Studies). HCA2 cells were isolated, in Dr. Smith’s laboratory, from neonatal fibroblasts (28). RNA preparation from precrisis, immortal, and 5-aza-dC–treated immortal MDAH041 and MDAH087 cells, CRL-1502 (PD 20–26), CCL-110 (PD 8), and senescing HCA2 (PD 75) is as described above. Total RNA (2 lg) was reverse transcribed into cDNA using SuperScript II (Invitrogen, Carlsbad, CA). Q-RT-PCR was performed using the SYBR Green PCR Detection Kit (PE Biosystems, Warrington, U.K.) and run on the ABI 5700 Sequence Detection System (Applied Biosystems, Foster City, CA). The Primer Express Program (Applied Biosystems) was used to design primers for Q-RT-PCR (Supplemental Table 7). [Supplemental Tables are linked to the online article in the September issue at http://biomed.gerontologyjournals.org/content/vol61/ issue9/.] The relative fold change of the gene of interest is calculated by normalizing it to an endogenous gene, GAPDH, and comparing it to a control sample, 2CT, where, CT ¼ (CT Gene of interest CT GAPDH)experiment

(CT Gene of interest CT GAPDH)control) (29). If the relative fold change was between 0 and 1, then the fold change was calculated by dividing 1 by the relative fold change. Fold changes of replicates from multiple independent experiments were averaged.

Gene Ontology Analysis We used GoMiner software (version 122; http:// discover.nci.nih.gov/gominer/index.jsp) (30) to annotate our gene expression data with Gene Ontology (GO) categories. The entire HGU95Av2 GeneChip probe set was the reference. We analyzed four experimental genes lists: genes that were up- and downregulated during immortalization in all four immortal LFS cell lines, and genes that were up- and downregulated after 5-aza-dC treatment in all four immortal LFS cell lines (Supplemental Table 1). The probes from the lists were first converted to unique gene symbols using NetAffx (http://www. affymetrix.com/), the Affymetrix online database (Build #166) (31), and then the unique list of gene symbols was analyzed by GoMiner. The 8487 unique gene symbols on the HGU95Av2 GeneChip were linked to 6020 GO categories. We used the one-sided Fisher’s exact test p values calculated by GoMiner to evaluate the statistical significance of changes for a GO category (Supplemental Figure 3). [Supplemental Figures are linked to the online article in the September issue at http://biomed. gerontologyjournals.org/content/vol61/issue9/.] FDR (false discovery rate) was used to correct for type I error for an individual test to achieve an acceptable type I error at the level of the whole experiment. FDR is one of the least conservative correction methods, and allows for tests of variables with some dependencies (32,33). The corrected p values were calculated as (P 3 R) / i) (P is the original p value calculated from GoMiner; i is the index for increasingsorted p value; R is the total GO categories) for each GO category. RESULTS In a preliminary study, we examined the gene expression changes during immortalization of the telomerase-positive LFS immortal cell line MDAH041 and the role of methylation-dependent gene silencing in that process. We found a statistically significant proportion of IFN genes dysregulated during immortalization (18). To examine the significance of the role of the IFN pathway and to identify other mechanisms commonly abrogated during immortalization, we used four independently immortalized LFS cell lines derived from the fibroblasts of two different LFS patients. The telomerase-positive cell line MDAH041, and the three MDAH087 telomerase-negative, ALT cell lines (8,10), MDAH087-N, MDAH087-1, and MDAH087-10, were used in a systematic analysis of the gene expression changes during immortalization and after 5-aza-dC treatment. To confirm that the immortal LFS cell lines resulted from independent immortalization events, we performed limited genotyping of key genes known to change during immortalization to (Supplemental Figure 1 and associated text).


MDAH087-1, and MDAH087-10 were each compared with two RNA preparations from MDAH087-PC cells, individually. Three RNA preparations from 5-aza-dC–treated MDAH087-N, MDAH087-1, and MDAH087-10 were each compared with RNA preparations from the corresponding untreated immortal MDAH087 cells separately. Microarray data on MDAH041-PC, MDAH041 immortal, and MDAH041 5-aza-dC–treated cells [from previous experiments in our laboratory (18)], were used in the microarray analysis performed in this study. We used the Affymetrix DMT version 5 to select genes with increased expression. Probes with a detection call of present or marginal (detection p value cutoffs, a1 ¼ .04 and a2 ¼ .06), and those that had a change call of increase or marginal increase (change p value, c1 ¼ .0025 and c2 ¼ .003) in all four immortal LFS cell lines, irrespective of fold change, in .65% of the chip comparisons, were identified as increased. Genes with decreased expression were similarly detected, but probes with a detection call of absent were also included.

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Genes Downregulated After Immortalization by Gene Methylation In our initial study of only one LFS cell line [MDAH041 (18)], 85 genes were repressed during immortalization and upregulated after treatment with 5-aza-dC. This finding

Table 1. Categorization of Genes Regulated in All Four Immortal Li-Fraumeni Syndrome (LFS) Cell Lines

Comparison A. IM vs PC upregulated B. IM vs PC downregulated C. 5-aza-dC vs IM downregulated D. 5-aza-dC vs IM upregulated Genes in B and D Genes in A and C

Common to Four IM IFN-Regulated p53-Regulated Imprinted Cell Lines Gene Gene Gene 149

43

29

0

187

40

23

4

46

10

7

0

185 14 2

56 2 1

26 1 1

0 0 0

Note: PC ¼ precrisis cells; IM ¼ immortal cells; 5-aza-dC ¼ 5-azadeoxycytidine (5-aza-dC)–treated immortal cells; IFN ¼ interferon.

indicated the significance of methylation-dependent gene silencing to the process of immortalization. In this study hierarchical clustering revealed that, of the genes that decreased during immortalization, 80% increased in at least one immortal LFS cell line following 5-aza-dC treatment (Supplemental Figure 2B). Furthermore, we identified 14 genes the expression of which decreased during immortalization and increased after treatment with 5-aza-dC that were common to all four immortal LFS cell lines (Table 2; Supplemental Table 1, genes in sets B and D). There is a significantly greater proportion of methylation-sensitive genes (14 genes) than genes upregulated during immortalization and upregulated after 5-aza-dC treatment (7 genes), genes upregulated during immortalization and downregulated after 5-aza-dC treatment (2 genes), or genes downregulated during immortalization and downregulated after 5-aza-dC treatment (4 genes) (Fisher’s exact test, p ¼ .01). Single nucleotide polymorphism analysis of the 14 genes showed that, in general, loss of gene expression during immortalization was not frequently associated with allelic loss, but rather other mechanisms (such as epigenetic silencing) are involved (data not shown). This finding is consistent with a comprehensive single nucleotide polymorphism analysis of the four LFS cell lines that indicated that alleleic loss in general was infrequent (data not shown). Many of the genes that increased during immortalization decreased after 5-aza-dC treatment (Supplemental Figure 2A). There were, however, only two genes identified that were upregulated during immortalization and decreased after 5-aza-dC treatment in all four immortal cells, cell division cycle 25B (CDC25B) and LIM domain-binding 2 (LDB2) (Supplemental Table 1, genes in sets A and C). That 14 methylation-sensitive genes increase during immortalization and only 2 genes decrease following 5-aza-dC treatment supports our preliminary observation (18) that methylation-dependent gene silencing is associated with the spontaneous immortalization of LFS cell lines.

Confirmation of Microarray Gene Expression Data by Q-RT-PCR Analysis To confirm the gene expression changes observed in our microarray data, Q-RT-PCR was used (Supplemental Table


Microarray Profiling of Gene Expression in Immortal LFS Fibroblasts In the microarray analysis, the immortal MDAH041 cell line was compared with the MDAH041-PC cell line and the immortal MDAH087 cell lines (MDAH087-N, MDAH0871, and MDAH087-10) were individually compared with the MDAH087-PC cell line. Hierarchical clustering of the differentially expressed genes is shown in Supplemental Figure 2. The number of genes differentially expressed during immortalization for each of the four LFS immortal cell lines is shown in Supplemental Table 1. Using this approach, we found 149 upregulated and 187 downregulated genes common to all four immortal LFS cell lines. Remarkably, although we did not specify a fold change, 98% of the changes in gene expression as detected by the microarrays were greater than 1.5-fold and 67% were greater than 2-fold. Because the immortal LFS cells do not express wild-type p53, we predicted that a significant proportion of genes with changes in expression would be p53 regulated. As predicted, of the 336 genes with a change in expression (149 upregulated and 187 downregulated), a significantly greater proportion of p53-regulated genes changed than was expected (16%) based on the total number of genes on the HGU95Av2 GeneChip (11%) the expression of which changed (p ¼ .01, Table 1 and Supplemental Table 2A). In addition, there were four known imprinted genes (PHLDA2, CD81, MEG3, and NDN) (Table 1) among the 187 downregulated genes, further indicating that methylation-dependent silencing is important to the mechanism of cellular immortalization. Treatment of LFS immortal fibroblasts with the DNMT inhibitor 5-aza-dC induces growth arrest and senescence (26). Previously, we showed that treatment of immortal MDAH041 cells with 5-aza-dC induced significant changes in gene expression (18). Immortal LFS cells treated with 5-aza-dC have a senescence-like morphology, and exhibited senescence-associated b-galactosidase activity (2,18,26). The number of genes differentially expressed following 5-aza-dC treatment for each of the four LFS immortal cell lines is shown in Supplemental Table 1. Among the four spontaneously immortalized LFS cell lines, 5-aza-dC treatment upregulated 185 genes and downregulated 46 genes in all four immortal LFS cell lines. None of these genes were known imprinted genes. Among the 33 p53regulated genes the expression of which changed following treatment with 5-aza-dC, there was a greater proportion that was upregulated than downregulated (79% and 21%, respectively; p ¼ .001) (Supplemental Table 2B). The proportion of 5-aza-dC-upregulated and -downregulated p53-regulated genes may be a reflection of the overall proportion of 5-aza-dC-induced gene expression changes (80% upregulated and 20% downregulated) (Table 1 and Supplemental Table 1).


883

Table 2. Fourteen Genes Downregulated After Immortalization and Upregulated by Demethylation in Li-Fraumeni Syndrome (LFS) Cells

Gene

Entrez Gene ID

MDAH041

MDAH087-N

MDAH087-1

MDAH087-10

5A

IM

5A

IM

5A

IM

5A

CpG

2.7

2.8

2.3

3.2

8.0

2.3

4.4

3.8

þ

CLTB CREG

1212 8804

1.3 1.6

1.5 2.2

1.9 1.5

2.4 1.6

4.0 9.3

2.1 4.1

2.4 3.3

1.8 2.6

þ þ

CYP1B1

1545

14.0

8.2

8.4

8.0

4.2

4.9

5.1

4.7

þ

84909 11234

2.7 1.6

3.8 1.6

6.0 1.6

2.3 2.2

2.0 1.7

1.8 1.7

2.2 2.4

1.7 1.5

þ þ

3306

2.0

1.6

12.4

10.9

5.2

5.3

5.2

6.4

HTATIP2

10553

17.7

5.6

6.6

3.7

2.9

2.0

5.5

2.4

þ

IGFBPrP1

3490

2.0

2.5

4.5

3.0

10.1

1.9

4.9

1.7

þ

KIAA1750 MAP1LC3B

85453 81631

27.8 1.6

2.9 1.7

18.8 2.5

5.1 2.5

3.7 2.0

3.3 3.1

15.2 1.3

9.1 1.8

þ þ

OPTN SERPINB2

10133 5055

3.0 1.3

2.6 4.2

1.8 3.0

1.8 10.7

3.6 2.6

1.6 8.8

2.1 4.3

1.3 7.0

þ

TNFAIP2

7127

6.0

5.4

9.3

5.4

4.3

3.1

3.7

2.1

FLJ14675 HPS5 HSPA2

Locus

15q26.3 Tumorigenesis: induced by p53 (34) IFN: regulated by IFNy 5q35 No related function 1q24 Cell cycle and tumorigenesis: promotes differentiation; overexpression delays G1/S transition and inhibits growth in human teratocarcinoma NTERA-2 cells (35) 2p21 Senescence: identified in normal human oral keratinocytes as a senescence-associated gene (36) 9q22 — 11p14 Cytoskeleton: gene also known as alpha integrin binding protein 63 14q24.1 Tumorigenesis: association of a polymorphism in HSPA2 with the susceptibility to the nasopharyngeal carcinoma (37) 11p15.1 Tumorigenesis: promotes apoptosis (38) Immortalization: cells from HTATIP2 knockout mice immortalize, whereas cells from HTATIP2 wild-type mice do not (H. Xiao, unpublished observations, 2004) 4q12 Tumorigenesis: regulated by p53 (M. Tainsky, unpublished observations, 2005). Downregulation associated with tumorigenesis (39) Cell cycle: downregulation IGFBPrP1 associated with downregulation of Rb and cyclin E (40) Senescence: expression increases during senescence (41); inhibits cell growth via a senescence-like mechanism (42) 8q22.1 — 16q24.2 Cytoskeleton: gene also known as microtubule-associated protein 1 light chain 3 beta 10p13 IFN: Regulated by IFN* 18q21.3 Senescence: overexpressed in senescent skin lung cells (43) and in senescent human mammary fibroblasts (44) IFN: Induces IFN-a/b production (45); interaction with Rb (46) 14q32 Tumorigenesis: disruption contributed to progression of cervical cancer (47)

Notes: Fold change in gene expression was determined using Affymetrix Data Mining Tool version 5. A representative probe is shown for genes with multiple probes. *Additional information can be found at http://www.karmanos.org/;tainskym/ y D. Leaman, unpublished observations. IM ¼ immortal cells versus precrisis cells; 5A ¼ 5-aza-deoxycytidine (5-aza-dC)–treated immortal cells versus untreated immortal cells; IFN ¼ interferon.

3). We examined the expression of 8 representative genes (ALDH1A3, CLTB, CREG, HSPA2, KIAA1750, OPTN, SERPINB2, and TNFAIP2) among the 14 genes that we identified as decreased during immortalization and increased after 5-aza-dC treatment in all four immortal LFS cell lines. Of the 14 genes that were methylation-sensitive during immortalization, 4 (CYP1B1, CLTB, HSPA2, and OPTN) had multiple probes on the Affymetrix HGU95Av2 GeneChip. Except for CYP1B1, all of the replicate probes on the chip for these genes were not identified as decreased during immortalization and increased after 5-aza-dC treatment in all four immortal LFS cell lines. Even so, Q-RTPCR did confirm that the genes with such discrepancies between their multiple probes were in fact methylationsensitive during immortalization. Overall, 99% (Supplemental Table 3, 71 of 72 comparisons) of the changes in gene expression observed in the microarrays were confirmed by Q-RT-PCR. We considered microarray and Q-RT-PCR fold changes in concordance when both had significant fold

changes in the same direction or when neither had a significant fold change. The cutoff for significant fold change for microarray was 61.3-fold, and for Q-RT-PCR was 61.8-fold. This confirmation indicates that microarrays are a reliable method to measure gene expression changes, and thus these data can be used in further analyses, such as identification of critical pathways.

Epigenetic Control of IFN-Regulated Genes in the LFS Cell Lines In our preliminary study, 39 of the 85 genes identified in MDAH041 as epigenetically repressed during immortalization were known IFN signaling pathway–regulated genes (18). The probability of that occurring by chance was ,.0001; thus we concluded that the IFN-pathway genes played a significant mechanistic role in the acquisition of cellular immortalization. Consistent with our preliminary study we found that a significantly greater proportion of IFN-regulated genes common to all four LFS immortal cell


IM

220

ALDH1A3

Function of Gene Related to Immortalization, Senescence, or Tumorigenesis, Including the Three Pathways Identified in This Article: IFN, Cell Cycle, Cytoskeleton*

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Identification of Pathways Associated With Cellular Immortalization To identify mechanisms (in addition to the IFN-regulated pathway) critical to cellular immortalization, we studied the biological, cellular, and molecular characteristics of the

genes differentially expressed during immortalization and after 5-aza-dC treatment (Supplemental Table 1) using the GO software, GoMiner. GoMiner links genes with GO categories, allowing us to identify the biological process, cellular component, and molecular function associated with the genes in our gene lists (30). The p value for each GO category was calculated as an evaluation of the significance of the number of gene changes associated with a GO category. Subcategories of the three primary GO categories (biological processes, cellular components, and molecular function), were plotted (Supplemental Figure 3) to evaluate which functional categories were significant during immortalization and/or following 5-aza-dC treatment. Sublayers of the significant GO categories were then explored to determine more specifically which GO functional categories accounted for the changes. To avoid misinterpretations resulting from failure to correct for multiple comparisons in GoMiner, we recalculated the p values using FDR (Supplemental Figure 3, denoted by asterisks) for each GO category. Possibly due to the small number (,200) of dysregulated genes in the lists, there were only a few GO categories that achieved significance after correction by FDR. In the primary GO category biological processes, among the categories with a significant number of genes upregulated during immortalization only cell proliferation (GO:0008283) remained significant after correction by FDR. Within the cell proliferation (GO:0008283) category, most of the changes occurred in cell cycle (GO:0007049), cytokinesis (GO:0000910), and regulation of cell proliferation (GO:0042172). Only cell cycle (GO:0007049) remained significant after correction by FDR. In these categories, MYC, E2F transcription factor 4 (E2F4), cyclindependent kinase inhibitor 1A (CDKN1A), and cyclindependent kinase inhibitor 3 (CDKN3) were among the genes identified as dysregulated during immortalization. After treatment of the cell lines with 5-aza-dC, the cell proliferation (GO:0008283) category had a significant number of genes, including cell division cycle 34 (CDC34), cyclin-dependent kinase inhibitor 2C (CDKN2C), and fibroblast growth factor 2 (FGF2), with changes in gene expression. It is interesting that, in the cell proliferation category (GO:0008283), among the 37 genes (Supplemental Table 4) the expression of which was upregulated during immortalization and the 11 genes the expression of which was downregulated following demethylation, only one gene (CDC25B) was found in both gene sets. Among the GO categories identified for genes differentially expressed after 5-aza-dC treatment, the only GO category that remained significant after correction by FDR was response to wounding (GO:009611), a subcategory of response to extracellular stimulus (GO:0009991). This finding is consistent with another of our findings that the IFN pathway is a significant pathway in immortalization (18), as 12 of the 19 genes identified in the wounding category (GO:009611) are IFN- and/or cytokine-regulated genes (Supplemental Table 5). In the primary GO category cellular component (Supplemental Figure 3B), we identified several subcategories that had a significant number of genes that were downregulated


lines (compared to the proportion on the HGU95Av2 GeneChip that ever changed in expression) were regulated during immortalization (p ¼ .01), and were regulated following 5-aza-dC treatment ( p ¼ .0008) (Supplemental Table 2A). Our working hypothesis was that genes decrease during immortalization by epigenetic silencing; therefore, we were interested in determining whether a greater proportion of genes decreased than increased during immortalization and whether a greater proportion increased than decreased following 5-aza-dC treatment. The difference between the proportion of IFN genes that increased and the proportion that decreased during immortalization is not statistically significant ( p ¼ .83, Supplemental Table 2B). As anticipated, a significantly greater proportion of genes that were common to all four LFS cell lines increased than decreased following 5-aza-dC treatment ( p , .0001 in all cases, Supplemental Table 2B), thus emphasizing the importance of gene silencing by methylation during the process of immortalization. Only 2 of the 14 methylationsensitive genes common to all four LFS immortal cell lines, however, are IFN-regulated (Table 1). These findings support the conclusion from our preliminary study with MDAH041 cells that epigenetic repression of IFN-regulated genes is significant during immortalization. Among the four immortal cell lines, however, different subsets of IFN genes are dysregulated. In the analysis of the individual immortal LFS cell lines, there was a statistically significant proportion of IFNregulated genes that were differentially expressed during immortalization ( p , .0001 in all cases, Supplemental Table 2A). In MDAH041, MDAH087-N, and MDAH087-1, there was a statistically significant difference between the proportion of genes that increased and decreased during immortalization (p ¼ .02, .003, and .01, respectively; Supplemental Table 2B); there was, however, a higher proportion of genes in MDAH041 with decreased expression, whereas in MDAH087-N and MDAH087-1, there was a higher proportion of genes with increased expression. There was no difference in the proportion of genes that increased and decreased during immortalization in MDAH087-10 (p ¼ .35, Supplemental Table 2B). The proportion of genes that increased following 5-aza-dC treatment was of marginal statistical significance in MDAH041 and MDAH087-N ( p ¼ .08 for each) and was statistically significant in MDAH087-10 ( p , .0001, Supplemental Table 2B). There was no difference in the proportion of genes that changed following 5-aza-dC treatment in MDAH087-1 (p ¼ .67, Supplemental Table 2B). We concluded that although the IFN pathway appears to play a significant role in the cellular immortalization of LFS cell lines, there are differences among the cell lines in the specific genes within this pathway that are dysregulated and in the mechanisms by which the IFN pathway is abrogated.


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regulation of cell proliferation category (GO:0008283), except for one, IGFBPrP1, which is one of the 14 methylation-sensitive downregulated genes. This finding indicates that the arrest of cell proliferation (GO:0008283) associated with 5-aza-dC treatment results in changes in a different set of genes from those altered that permit cells to maintain their proliferative capacity during immortalization.

during immortalization, but only the GO categories nucleus (GO:0005634) and cytoskeleton (GO:0005856) remained significant after correction by FDR. After treatment with 5aza-dC there was a significant number of upregulated genes, even after correction by FDR, in the GO category chromosome (GO:0005694). No subcategories of cellular component had a significant number of genes that decreased after 5-aza-dC treatment. In the primary GO category molecular function (Supplemental Figure 3C), there were no GO categories that retained significance after correction by FDR. There was, however, a significant number of genes in the subcategories cell adhesion molecule activity (GO:0005194) and structural molecule activity (GO:0005198) that were downregulated during immortalization; this finding is consistent with cytoskeleton genes (GO:0005856) being identified as significant in the cellular component. Eleven of the 19 genes from the category structural molecular activity (GO:0005198) and 1 of the 9 genes from the category cell adhesion molecular activity (GO:0005194) overlap with the genes in the cytoskeletal category (GO:0005856) (Supplemental Table 6). Additionally, by western blot analysis we found that the cytoskeletal protein gelsolin was methylationsensitive in MDAH041 immortal cells (data not shown). There were two GO categories examined that were both significantly downregulated during immortalization and upregulated during demethylation: (i) a subcategory of biological process, regulation of cell proliferation (GO:0042127, p value Immortal Down , .02; p value 5aza-dC Up , .05) and (ii) a subcategory of cellular component, extracellular (GO:0005576, p value Immortal Down , .005; p value 5-aza-dC Up , .01). The genes with reduced expression after immortalization did not overlap with the genes upregulated during demethylation in the


Figure 1. Virus sensitivity of the four immortal Li-Fraumeni syndrome (LFS) cell lines. Precrisis and immortal LFS cells were evaluated for the ability to induce an interferon (IFN)-a– and b–induced antiviral response to vesicular stomatitis virus (VSV) at a low (0.05 pfu/mL) multiplicity of infection. Results are expressed as the means 6 standard deviation from at least two independent experiments (n ¼ 8). The values are all normalized to control uninfected cells that were set to 0% (100% cell viability). AFB ¼ normal diploid human fibroblasts; 041-PC ¼ precrisis MDAH041; 041-IM ¼ immortal MDAH041; 087-PC ¼ precrisis MDAH087; 087-N ¼ MDAH087-N; 087-1 ¼ MDAH087-1; 087-10 ¼ MDAH087-10 cells.

Analysis of the Three Pathways Identified That Are Altered in Cellular Immortalization In our analysis of the microarray data, we identified three pathways that are altered in cellular immortalization of LFS cells: cell cycle, cytoskeletal, and IFN pathway. The demonstration that the cell-cycle genes p16INK4A and p21CIP1/WAF1 could induce senescence when expressed in MDAH041 and MDAH087-N (26) in a sense verified our identification of cell cycle as an important pathway. Given the known disruption of cytoskeletal organization in immortalization (48), our observation of dysregulation of cytoskeletal genes during cellular immortalization was consistent with this well-known phenomenon and with typical visual inspection of the variations of morphology in immortal and senescent LFS cells. The IFN pathway was the third pathway that we identified as altered during cellular immortalization. To further evaluate the involvement of the IFN pathway in immortalization, we determined the expression of the IFN signaling pathway gene, STAT1a, by western blot analysis (Supplemental Figure 1). Consistent with STAT1a transcript expression (Supplemental Table 3), protein levels of STAT1a decreased in immortal MDAH041 cells and increased in response to treatment of immortal MDAH041 cells with 5-aza-dC. Changes in STAT1a protein and mRNA expression were also analyzed in MDAH087 cells. As was seen in MDAH041, STAT1a gene expression decreased during immortalization and increased after 5-azadC treatment in MDAH087 cells. It is interesting that, in contrast to Q-RT-PCR and microarray data, there was little to no difference in STAT1a protein expression levels between MDAH087-PC and the immortal MDAH087 cell lines, nor was there a significant difference between untreated and 5-aza-dC–treated cell lines. Infection with the vesicular stomatitis virus (VSV) can activate the IFN pathway. VSV induces a cytolytic response in cells in which the IFN pathway is abrogated (49). We therefore performed, by using VSV viral sensitivity assays, a functional analysis of the IFN pathway after immortalization in the LFS cell lines. This assay assesses the innate immunity that the IFN pathway provides to cells. Cells that have a fully functional antiviral IFN system are able to protect themselves against a low dose of virus. We observed an increase in cell lysis in immortal MDAH087-N, MDAH087-1, MDAH087-10, and MDAH041 cells compared to the respective precrisis cells after VSV infection (Figure 1). The addition of exogenous IFN-a to the culture medium restored the innate immunity as indicated by loss of viral sensitivity to 3 of 4 of these immortal cell lines (data not shown). In addition, poly (I:C) treatment induced STAT1a, its phosphorylation, and IFNb gene expression in MDAH041-PC, MDAH041-IM, MDAH087-PC, and

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Table 3. Q-RT-PCR Analysis of the Combined Effects of IFN-a and 5-aza-dC on Genes in MDAH041 Cells Control vs 5-aza-dC

Control vs 5-aza-dC þ IFN-a

Control vs IFN-a

5-aza-dC vs 5-aza-dC þ IFN-a

Gene

PC

IM

PC

IM

PC

IM

PC

IM

IFN-a IFN-b IFN-regulatory factor 7 (IRF7)

2 1 2.4

2.4 8.5 8.6

1 8 7

1 86 1

1.3 9.8 45

2.5 445 4534

2.5 9.3 18.9

1.3 52 528

Note: Q-RT-PCR ¼ real-time reverse transcription polymerase chain reaction; IFN ¼ interferon; PC ¼ precrisis cells; IM ¼ immortal cells; 5-aza-dC ¼ 5-azadeoxycytidine (5-aza-dC)-treated cells.

Table 5. Comparison of Expression Levels of IFN-Regulated Genes Using Q-RT-PCR: Upregulation in Senescing Normal Diploid Fibroblasts as Compared to Two Strains of Young Normal Diploid Fibroblasts

Table 4. Q-RT-PCR Analysis of the Effect of IFN-a on IFN-Regulated Genes in MDAH041 Cells MDAH041-PC Gene AIM2 CIG49 IFN-induced 17-kd/15-kd protein (IFI15) IFN-inducible protein 9-27 (IFITM1) IFNa-inducible p27 IFN-inducible protein p78, (Mx1) Gamma-IFN-inducible protein (IP30) IFN-regulatory factor 7 (IRF7) OAS1 OAS2 IFN-inducible 56 kd protein (IFI56) STAT1a

MDAH041-IM

IFN-a 8 Hours

IFN-a 30 Hours

IFN-a 8 Hours

IFN-a 30 Hours

4.7 5

4.7 3

1.3 261

7.2 601

1.2

1.1

111

568

21 43

6353 2282

2.6 5

13 27

enhancement of IRF7 and IFN-b expression (Table 3). Thus, inhibition of IRF7 expression may be a critical epigenetic step leading to cellular immortalization. These data further support that the IFN pathway is dysregulated in immortalization and that IRF7 may be a key regulator of this process. To further assess if the IFN pathway is associated with senescence, we compared the expression of known IFNregulated genes in two strains of normal diploid fibroblasts with gene expression in RNAs from normal diploid fibroblasts at the time of senescence and found that most (10/12) of these genes are at least 3-fold upregulated at senescence in one or more comparisons (Table 5). The upregulation of these genes in RNA from senescing cells is consistent with a requirement for downregulation of IFN-regulated genes for immortalization to occur. Therefore, the upregulation of IFN-regulated genes in senescing cells is probably the strongest indication that the downregulation of the IFN pathway is mechanistically important to achieving immortalization. Indeed, senescing human fibroblasts produce 28-fold more IFN-a and 10-fold more IFN-b RNA than do their young cell counterparts (data not shown).

5

5.5

17

834

2 2 7 5

1.9 1.8 11 4

1.7 1 749 5595

6 1 4162 9047

6.2 13.5

10 14.5

1267 43

2352 120

Note: Q-RT-PCR ¼ real-time reverse transcription polymerase chain reaction; IFN ¼ interferon; PC ¼ precrisis cells; IM ¼ immortal cells.

Gene AIM2 CIG49 IFN-induced 17-kd/15-kd protein (IFI15) IFN-inducible protein 9-27 (IFITM1) IFN-a-inducible p27 IFN-inducible protein p78 (Mx1) Gamma-IFN-inducible protein (IP30) IFN-regulatory factor 7 (IRF7) OAS1 OAS2 IFN-inducible 56 kd protein (IFI56) STAT1-a

SEN vs CRL-1502 (1)

SEN vs CRL-1502 (2)

SEN vs CCL-110

1.7 11.3

1.2 11.4

43 7.3

1.2

1.3

1.1

10.2 1.6 11.5

53 1.8 27

43 1.2 26

64

29

2.1 753 6

2.0 3500 19

3.3 67

4.5 63

1.7 7.8 188 17 14 28

Notes: Normal diploid fibroblasts: CRL1502 (two independent RNA preparations) and CCL110. IFN ¼ interferon; Q-RT-PCR ¼ real-time reverse transcription polymerase chain reaction; SEN ¼ senescing normal diploid fibroblasts, HCA2 cells.


MDAH087-N, but there was no induction of these changes in either MDAH087-1 or MDAH087-10 (data not shown). Thus the VSV and poly (I:C) viral sensitivity assays functionally demonstrate that the IFN pathway is dysregulated in LFS cells after immortalization. The fact that the viral sensitivity results were not identical for each of the cell lines indicates that the mechanism by which the IFN pathway is abrogated probably differs among the immortal cell lines. Treatment of immortal MDAH041, but not MDAH041PC cells with 5-aza-dC induces expression of IFN-b (Table 3). That 5-aza-dC is able to induce the expression of IFN in immortal cells, but not precrisis cells, is further proof that the IFN pathway is disrupted during immortalization. In addition, this finding suggests that restoration of the IFN pathway following 5-aza-dC treatment contributes to cells senescing following 5-aza-dC treatment. Furthermore, when we treat immortal MDAH041 cells with IFN-a, IFNregulated genes are upregulated (Table 4). The IFNinducible transcription factor IFN regulatory factor 7 (IRF7), which is required for the second wave of gene induction by IFNs (50), was not, however, inducible by IFN-a. In contrast, IRF7 was inducible by 5-aza-dC. Treatment of immortal, but not precrisis MDAH041 cells with IFN-a in combination with 5-aza-dC led to synergistic


that IFN signaling pathways were silenced by methylation in a preneoplastic step of cancer development, immortalization (18). These results supported our hypothesis that IFN signaling pathway genes, in particular IRF7, may act as growth suppressors in the progression of cells to immortalization. The reduced expression of IFN-a may also contribute to immortalization and is likely a consequence of the loss of IRF7 expression (Q. Li, L. Tang, P. C. Roberts, J. M. Kraniak, O. Tehrani, and M. Tainsky, unpublished observations, 2005). By categorizing differentially expressed genes into GO categories, we determined that genes coding for regulatory proteins of the cell cycle and/or structural proteins of the cytoskeleton were differentially expressed during immortalization. Identification of the cell cycle as a significant pathway is consistent with studies that found that the cell cycle is dysregulated during immortalization (26,57). Specifically, our data support the observation that dysregulation of the cell cycle, through the retinoblastoma signaling pathway, and dysregulation of cyclin-dependent kinase inhibitors, is required for immortalization of fibroblasts (58,59). Because we were able to identify dysregulation of cell-cycle pathway genes using microarray, this finding validates our use of microarray as a tool for identifying additional cell-cycle genes and genes in other pathways not previously implicated in cellular immortalization. In the cell-cycle pathway, in addition to the well-known cell-cycle genes RB and p16INK4 that are involved in cellular immortalization, we found other cell-cycle regulators among the 14 methylation sensitive genes altered in all four immortal LFS cell lines. Four of these 14 genes regulate the cell cycle by being functionally associated with p53 or RB. IGFBPrP1 and ALDH1A3 are regulated by p53, and CREG and SERPINB2 physically associate with RB. In addition, CDC25B, which is increased in expression after immortalization and decreased in expression after 5-aza-dC treatment, is also regulated by p53 (M. Tainsky, unpublished observations, 2005). SERPINB2, which decreases during immortalization and increases after 5-aza-dC treatment, was found to be overexpressed in senescent skin cells (43) and senescent human mammary fibroblasts (44). In keeping with our hypothesis that the IFN pathway plays a key role in cellular immortalization, SERPINB2 is known to protect cells from alpha virus infection through the induction of IFNstimulated gene factor 3 and through the induction of lowlevel IFN-a and b production (45). We also report here the dysregulation of cytoskeletal genes in immortalization. That the cytoskeleton (GO:0005856) category is identified during bioinformatics analysis is consistent with the morphological changes that occur as cells senesce. Typically, as cells senesce they become very large and flat in comparison to immortal cells. Thus, one would predict that changes in cytoskeletal genes would contribute to the processes of immortalization and senescence (48). Overexpression of the cytoskeletal protein vimentin induced a senescent cell morphology in human fibroblasts (60). Furthermore, when the cytoskeletal gene cavelonin-1 was knocked out in senescent human diploid fibroblasts, the characteristic senescent cell morphology of the cells was


DISCUSSION Our goal in this study was to investigate epigenetic control of immortalization using four independent immortal cell lines derived from two different LFS patients. In our analysis, we identified several pathways with changes in gene expression, including the IFN signaling pathway, the cell-cycle pathway, and genes encoding cytoskeletal proteins. Fourteen genes were consistently methylationsensitive after immortalization in all the immortal cell lines studied. The gene expression profiles among the three immortal MDAH087-derived cell lines were more closely related to one another than MDAH041 was to any of these cell lines (Supplemental Figures 2 and 4). Although the GO pathways that were consistently altered among these cells were similar, the gene expression patterns were closely related, but not identical. Thus, we demonstrated that certain pathways, but not necessarily particular genes, must be abrogated or enhanced for a cell to become immortal. Furthermore, the mechanisms by which any particular pathway was disrupted varied among these cell lines. A significant number of the methylation-sensitive genes, in each of the four immortal LFS cell lines, were in the IFN pathway. Functional analysis of the IFN pathway in the LFS cell lines using poly (I:C) treatment and VSV viral sensitivity assays corroborated the abrogation of the IFN pathway in immortalization. After stimulation of the IFN pathway in LFS cells with either VSV infection or the double-strand RNA analog poly (I:C), we found that all four immortal cell lines were more sensitive to these treatments than were their respective precrisis cell lines or normal fibroblasts. Because different sets of IFN-regulated genes were dysregulated in each of the four immortal LFS cell lines, we concluded that abrogation of this pathway is necessary, albeit not sufficient, for immortalization to occur. However, the specific mechanism of inactivation of the IFN pathway varies from cell line to cell line. Additionally, because 5-aza-dC is able to induce the expression of IFN-regulated genes in immortal cells but not in precrisis cells, we hypothesized that restoration of the IFN pathway following 5-aza-dC treatment contributes to the cellular senescence observed following 5-aza-dC treatment. Specifically, our evidence suggests that inhibition of IRF7 expression may be a critical epigenetic step leading to cellular immortalization. The involvement of the IFN pathway in cellular senescence and tumorigenesis was further supported by the fact that a number of IFN-induced proteins, including double-stranded RNA activated protein kinase (PKR), activated RNaseL, and the 200 gene family, have tumor suppression activity when overexpressed in tumor cells (51). Increased expression of the IFN inducible gene, IFI16, was recently shown to contribute to induction of senescence in prostate epithelial and fibroblast cells (52,53). Studies examining promoter methylation in bladder cancer cells (54), colon tumor cells (55), and (most recently) pancreatic cancer cells (56) also showed that IFN signaling pathways were activated following treatment of cancer cells with 5aza-dC, thus implying that their promoters are silenced by DNA methylation. Our data were the first to demonstrate

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Summary We have used gene expression analysis using microarrays to identify pathways critical to the process of cellular immortalization. We found that the senescence-initiating events that led to genomic instability and telomere stabilization include the loss of checkpoint proteins such as p53, p21CIP1/WAF1, and p16INK4A. In addition, gene profiling revealed 149 upregulated genes and 187 downregulated genes, 14 of which were epigenetically downregulated in all four immortal LFS cell lines. We found several common pathways involved in immortalization including the IFN pathway, genes involved in proliferation and cell-cycle control, and genes encoding cytoskeletal proteins. ACKNOWLEDGMENTS This work was supported by the Barbara and Fred Erb Endowed Chair in Cancer Genetics (to M.A.T.), and by funds from the Barbara Ann Karmanos Cancer Institute, the State of Michigan Life Sciences Corridor, Wayne State University and Michigan Center for Genomic Technologies Applied Genomics Technology Center (MEDC 085P10009816), and the Genomics and Biostatistics Cores of the Barbara Ann Karmanos Cancer Institute (P30CA022453). We thank Drs. George Brush, Sylvia Dryden, Janice Kraniak, and Gen Sheng Wu for critical reading of the manuscript, and Scott Tainsky for help with bioinformatics analysis. We are grateful to Dr. Douglas Leaman, University of Toledo, for providing additional lists of IFN-regulated genes and to Dr. James Smith, University of Texas Health Science Center, San Antonio, for providing us with senescing HCA2 cells for the preparation of RNA. Drs. Fridman and Tang contributed equally to this work. Olga I. Kulaeva is now with the Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Piscataway.

Supplemental Figures and Tables are linked to the online article in the September issue at http://biomed.gerontologyjournals.org/content/ vol61/issue9/. For additional supporting text, figures, and tables for this article, see Michael A. Tainsky's Homepage at: http://www.karmanos.org/ ;tainskym/ Address correspondence to Michael A. Tainsky, PhD, Program in Molecular Biology and Human Genetics, Barbara Ann Karmanos Cancer Institute, and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, 110 East Warren Ave., Detroit, Michigan, 48201. E-mail: [email protected]

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disrupted, and the cells adopted the morphology of a young cell with a small spindle shape (61). Two of the 14 methylation-sensitive genes, MAP1LC3B and HPS5, are associated with the cytoskeleton. We also found that the cytoskeletal protein CRP1, which is regulated by IFN, decreases during immortalization in all four immortal LFS cell lines (data not shown), further supporting the involvement of the IFN pathway and cytoskeletal proteins in immortalization. We were surprised to find that, among the 14 methylation-sensitive genes that are common to all four immortal LFS cell lines, there was not a significantly higher proportion with CpG islands when compared to a set of 16 genes that were downregulated in immortal cells but not regulated by 5-aza-dC (data not shown). In addition, we did not find that the size of the CpG island(s) within the methylation-sensitive genes correlated with the gene being regulated by DNA demethylation. Of these 14 genes, there are 12 genes that have a known function, and 10 of these, ALDH1A3, CREG, HPS5, HSPA2, HTATIP2, IGFBPrP1, CYP1B1, MAP1LC3B, SERPINB2, also known as PAI-2, and TNFAIP2, have been associated with tumorigenesis, senescence, CpG methylation, cell cycle, or the cytoskeleton, and based on microarray analysis and literature (45,62) (D. Leaman, unpublished observations, 2003). Three of these genes have been shown to be regulated by IFN: ALDH1A3, OPTN, and SERPINB2 (Table 2).


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