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Ann Thorac Surg 2001;71:295-302
© 2001 The Society of Thoracic Surgeons

Original article: general thoracic

Induction of MAGE-3 expression in lung and esophageal cancer cells

Todd S. Weiser, MDa, Galen A. Ohnmacht, MDa, Zong S. Guo, PhDa, Maria R. Fischette, DOa, Gustavio A. Chen, MSa, Julie A. Hong, MSa, Dao M. Nguyen, MDa, David S. Schrump, MDa

a Thoracic Oncology Section, Surgery Branch, National Cancer Institute, Bethesda, Maryland, USA

Address reprint requests to Dr Schrump, Thoracic Oncology Section, Surgery Branch, National Cancer Institute, Building 10, Room 2B-07, 10 Center Dr, Bethesda, MD 20892-1502
e-mail: david_schrump{at}nih.gov

Presented at the Thirty-sixth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 31–Feb 2, 2000.


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Discussion
 References
 
Background. Although MAGE-3 has been detected in approximately 40% of lung and esophageal cancers, expression of this cancer testis antigen appears to be below the threshold for immune recognition in patients with these malignancies. The aim of this study was to determine if the demethylating agent, 5-Aza-2'-deoxycytidine (DAC) and if the histone deacetylase inhibitor Depsipeptide FR901228 (DP) could enhance MAGE-3 expression in lung and esophageal cancer cells.

Methods. Eleven lung and esophageal cancer lines and cultured normal human bronchial epithelial (NHBE) cells were exposed to normal media (NM), DAC, DP, or combination DAC/DP at varying concentrations and exposure durations. MAGE-3 expression was evaluated by quantitative RT-PCR (TaqMan) and immunohistochemistry techniques. Trypan blue exclusion techniques were used to examine the proliferation of cancer cells after drug exposure.

Results. Relative to untreated controls, MAGE-3 expression was enhanced 32-fold (range 3.9 to 110) by DAC alone (0.1 µmol/L x 72 h), 2.1-fold (0.4 to 4.2) by DP alone (25 ng/mL x 6h), and 57-fold (4.6 to 209) by sequential DAC/DP exposure. Increased MAGE-3 mRNA copy numbers coincided with enhanced protein levels in these cells. MAGE-3 expression persisted after drug exposure. Flow cytometry confirmed the presence of functional HLA class I expression in these cells. Sequential DAC/DP treatment mediated pronounced growth inhibition in cancer cells but not NHBE.

Conclusions. Sequential DAC/DP treatment may be a novel strategy to simultaneously augment MAGE-3 expression and induce growth arrest in thoracic malignancies.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Discussion
 References
 
Lung and esophageal cancers presently account for more than 1,000,000 cancers diagnosed each year throughout the world. The overall 5-year survival rate for patients with these neoplasms is less than 10% despite aggressive multimodality intervention. These data highlight the urgent need for a more fundamental appreciation of the mechanisms of aerodigestive tract carcinogenesis, and the identification of novel compounds that simultaneously augment the immunogenicity and inhibit the proliferation of lung and esophageal cancer cells [1, 2].

The MAGE-3 gene was initially identified by Gaugler and associates [3] following analysis of CTL reactivity against autologous melanoma cells. MAGE-3 belongs to a growing class of T-cell defined cancer testis antigens (CTA) that are normally expressed only in the testes and ovary, yet are aberrantly expressed in a wide variety of primary tumors and cancer lines [4]. Because testicular cells do not express HLA class I molecules [5], these cells cannot present MAGE-3 for recognition by cytolytic T cells. Hence, MAGE-3 represents a bona fide shared tumor antigen that may be exploited for cancer immunotherapy; peptide sequences derived from MAGE-3 have been reported to mediate regression of melanomas in preliminary clinical trials [6, 7].

Although nearly 75% of small cell lung cancers (SCLC) and approximately 40% of non–small cell lung cancers (NSCLC) and esophageal carcinomas express MAGE-3 [8–10], recognition of this cancer testis antigen in patients with these neoplasms appears to be limited. Whereas deficiencies regarding antigen processing could account for the lack of immune response to MAGE-3 in some of these patients [11], in many others levels of MAGE-3 expression may simply be below the threshold for immune recognition. Conceivably pharmacologic agents that augment the expression of MAGE-3 may enhance the immunogenicity of lung and esophageal cancers.

Global alterations in chromatin structure occur early during multistep carcinogenesis, profoundly influencing the expression and function of a wide variety of genes [12]. Under normal conditions, transcriptional regulation involves highly complex and interrelated processes including DNA methylation and histone deacetylation [13]. Briefly, methylation of cytosine nucleotides in promoter regions inhibits transcription and deacetylation of lysine residues in histone proteins further silences gene expression by facilitating chromatin condensation, rendering DNA inaccessible to the transcription machinery [14].

Genome-wide demethylation occurring in the context of malignant transformation facilitates the expression of several CTA including MAGE-1 and NY-ESO-1 [15, 16]; interestingly, despite genomic demethylation, tumor suppressor genes such as p16 are selectively silenced by promoter hypermethylation mechanisms [17]. Recent studies have demonstrated that the demethylating agent 5-Aza 2'deoxyazacytidine (DAC) can induce CTA and tumor suppressor gene expression in cancer cells [16, 18]. Additional experiments have revealed that histone deacetylase (HDAC) inhibitors such as trichostatin A can enhance DAC-mediated induction of tumor suppressor genes in these cells [19]. To date, the effects of HDAC inhibitors regarding augmentation of DAC-mediated induction of CTA have not been reported.

In the present study we sought to evaluate whether 5-Aza-2'-deoxycytidine and the HDAC inhibitor Depsipeptide FR901228 (DP) could enhance MAGE-3 expression in lung and esophageal cancer cells under conditions potentially achievable in clinical settings. We also sought to determine whether these global agents exhibited antiproliferative effects in cancer cells under these conditions. Data reported herein support further evaluation of sequential DAC/DP treatment as a means to simultaneously enhance CTA expression and induce growth arrest in thoracic malignancies.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Discussion
 References
 
Cell lines
Human non–small cell lung carcinoma (NSCLC) cell lines H-596, H-1650, H-2087, and H-2228, the squamous cell esophageal cancer lines TE-1, -2, -3, -12, and -13, as well as the melanoma cell line, 586 mel, were available in tissue culture banks at the National Cancer Institute. The esophageal adenocarcinoma cell lines BE-3 and SKGT-5 were provided by N. Altorki, Cornell University Medical College, New York, NY. These cell lines were maintained in RPMI 1640 containing 10% fetal bovine serum, glutamine, and antibiotics. Normal human bronchial epithelial (NHBE) cells were purchased from Clonetics, Inc (Walkersville, MD) and maintained in bronchial epithelial cell basal media (Clonetics, Inc).

RNA extraction and real-time quantitative RT-PCR analysis
Total RNA was isolated from cell lines using reagents and protocols provided in the RNeasy Mini Kit (Qiagen, Valencia, CA). Synthesis of cDNA was performed with 2 µg of total RNA using a reverse transcription kit (Promega, Madison, WI) and oligo (dT)15 primers. Quantitative RT-PCR (TaqMan) analysis of gene expression was performed in a blinded manner using an ABI prism 7700 Sequence Detection System (Perkin-Elmer, Foster City, CA) [20, 21] Primers and TaqMan probes were purchased from Custom Oligonucleotide Factory (Foster City, CA). TaqMan probes were labeled at the 5'-end with the reporter dye molecule FAM (6-carboxyfluorescein) and at the 3'-end with the quencher dye molecule TAMARA (6-carboxytetramethylrhodamine). MAGE-3 and ß-actin primer sequences have been published previously [22, 23]. Protocols for the generation of cDNA standards, PCR reactions, and thermal cycle parameters have been previously described [23]. Briefly, for each experimental sample, amplification curves were produced and the copy number for the gene of interest was determined by extrapolation from standard curves; cDNA from 586-mel was used to generate the standard curves for MAGE-3 and ß-actin. Standardization of samples was achieved by dividing the copy number of the target gene by that of the endogenous reference gene, ß-actin.

For semiquantitative RT-PCR experiments, cDNA was amplified using primers specific for MAGE-3 and GAPDH as previously described [16, 24]. Amplification was performed for 35 cycles at 94°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute. PCR products were resolved on 1.5% agarose gel and visualized by ethidium bromide staining.

DAC/DP exposure and proliferation assays
5-Aza-2'-deoxycytidine was purchased from Sigma (Sigma, St. Louis, MO). Depsipeptide FR901228 was obtained from the Investigational Drug Branch of the National Cancer Institute, Bethesda, MD. The effects of DAC, DP, or sequential DAC/DP treatment on MAGE-3 expression, as well as proliferation of cultured cells were determined after exposure to (1) normal media (NM) for 96 hours; (2) DAC 0.1 µmol/L for 72 hours, followed by NM for 24 hours; (3) NM for 72 hours, then DP 25 ng/mL for 6 hours, followed by NM for 18 hours; or (4) sequential DAC/DP, followed by NM for 18 hours. For each experiment day 0 represented the time when cells were initially exposed to either NM or DAC. During this treatment regimen the media were changed every day, and after appropriate drug exposure cells were washed with HBSS. Quantitative RT-PCR, and conventional RT-PCR analysis of gene expression were performed at the 96-hour time point of each experiment unless otherwise indicated. For proliferation assays, cells were exposed in triplicate to NM, DAC, DP, and sequential DAC/DP as described, and were harvested on days 3, 4, and 5. Viable cells were enumerated using trypan blue exclusion techniques.

Immunohistochemistry analysis of MAGE-3 expression
MAGE-3 protein expression in cancer cells was evaluated by immunoperoxidase techniques using the murine monoclonal antibody 57B (mAb-57B) generously provided by Dr G. Spagnoli (University of Basel, Basel, Switzerland) [25]. Briefly, tissue sections from formalin-fixed, paraffin-embedded cell blocks underwent antigen retrieval by means of an autoclave (121°C for 5 minutes), in low concentration citrate buffer (pH 6.0), and quenching of endogenous peroxidase with 3% hydrogen peroxide for 30 minutes. Tissue sections were incubated for 1 hour with mAb-57B (dilution 1:50) at room temperature. Detection of the primary antibody was achieved using reagents and protocols contained in the avidin-biotin horseradish peroxidase kit from Vector Laboratories, Burlingame, CA). Slides were then counterstained with hematoxylin and mounted for subsequent analysis.

Flow cytometry analysis
HLA class I expression in cultured cancer cells was determined by flow cytometry techniques using the W6/32 (Sera-Lab, Sussex, England) antibody, which recognizes only functional class I molecules and antibodies specific for the HLA phenotype of the cells as determined by PCR analysis of genomic DNA in the HLA Laboratory at the National Institutes of Health [16].

Statistical analysis
Data from proliferation assays were expressed as means ± standard deviations. One-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test was used for statistical analysis using Prism 2.0 software (Graphpad Software, Inc, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Discussion
 References
 
Previously published studies have provided the preclinical rationale for an ongoing phase I study at the National Cancer Institute examining the feasibility of using a continuous 72-hour DAC infusion to induce tumor antigen expression in patients with carcinomas involving the lung, esophagus, and pleura [16]. Because the maximal tolerable plasma concentration in this trial is not expected to exceed 0.1 µmol/L, we have conducted a series of experiments to determine the magnitude of target gene induction in cultured cancer cells at this DAC concentration and to ascertain whether DP could augment gene expression. Quantitative RT-PCR experiments, which are presented in detail in another manuscript (Weiser and colleagues, submitted for publication), enabled us to chose the following treatment regimens for further analysis: DAC 0.1 µmol/L x 72 h (to reflect the exposure achievable at the MTD in our current trial), DP 25 ng/mL x 6 h (concentration and exposure duration compatible with those observed at the MTD during a recent unpublished phase I study at the NCI), and sequential DAC (0.1 µmol/L x 72 h)/DP (25 ng/mL) exposure. Depsipeptide doses exceeding 25 ng/mL and sequential DP/DAC regimens proved too toxic to allow quantitation of target gene induction.

The effects of DAC or DP alone or of sequential DAC/DP treatment were examined in a panel of lung and esophageal cancer cells exhibiting functional HLA class I expression (data available upon request) and different levels of MAGE-3 mRNA in the basal state. Representative quantitative RT-PCR data are summarized in Table 1. Variable induction of MAGE-3 antigen expression was observed in lung cancer cell lines after DAC exposure, ranging from 3.2-fold induction in H-2087 cell lines that normally have moderate levels of MAGE-3 transcripts, to 100-fold induction in H-2228 lung cancer cells in which MAGE-3 expression is extremely low in the basal state. Depsipeptide alone resulted in an average 2.3-fold induction of MAGE-3 (range 0.9 to 3.5) relative to basal expression. Sequential DAC (0.1 µmol/L x 72 h)/DP (25 ng/mL x 6 h) exposure mediated a substantial increase in MAGE-3 mRNA copy number in cell lines in which basal expression of this CTA was low (average 68-fold, range 30 to 141). Interestingly, sequential drug treatment increased MAGE-3 copy number to a lesser extent (4-fold) in H-2087 cells expressing relatively moderate levels of MAGE-3 mRNA in the basal state.


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  		Table 1. Quantitative RT-PCR Analysis of MAGE-3 Expression in Cancer Cells

 
Similar findings were noted after treatment of esophageal carcinoma lines. DAC treatment alone induced an average 52-fold induction in MAGE-3 expression in four of the seven esophageal carcinomas (TE-1, TE-13, SKGT-5, and BE-3), whereas in the remaining three lines the magnitude of gene induction was considerably less (average 5.7-fold). As observed in lung cancer cells, DP mediated an approximately 2-fold induction of MAGE-3 antigen expression (range 0.4 to 4.2) in the esophageal cancer lines. Interestingly, the magnitude of MAGE-3 induction appeared to be consistently higher in the four cell lines that were more responsive to DAC (average fold induction 3.5, range 2 to 4.2) relative to that observed in the three lines in which DAC mediated less MAGE-3 induction (average fold induction 0.47, range 0.37 to 0.45). Sequential DAC/DP exposure synergistically enhanced MAGE-3 copy number relative to basal expression in all of the esophageal lines examined; once again, the magnitude of MAGE-3 induction was most pronounced in those cell lines which were more responsive to DAC treatment. Nevertheless, MAGE-3 copy number exceeded 1000 per 104 copies of ß-actin in all of these lines after sequential drug exposure. Although some variability regarding the magnitude of gene induction was noted in individual cell lines during different quantitative RT-PCR experiments, sequential DAC/DP treatment reproducibly enhanced MAGE-3 expression to a greater extent than that observed after exposure to DAC or DP alone. Similar treatment had no significant effects regarding induction of MAGE-3 expression in cultured NHBE cells.

In subsequent experiments, conventional semiquantitative RT-PCR techniques were used to examine the magnitude of MAGE-3 gene induction detected by TaqMan methods. A representative experiment pertaining to TE-1 and TE-13 esophageal cancer lines is shown in Figure 1. Levels of MAGE-3 expression were undetectable by conventional RT-PCR techniques in either cell line exposed to normal media, or DP alone. After exposure to DAC, expression of MAGE-3 could be readily detected only in the TE-1 cells by standard RT-PCR techniques. MAGE-3 expression was clearly enhanced in TE-1 and TE-13 cells after sequential DAC/DP exposure. Results of these semiquantitative RT-PCR experiments confirmed those observed following TaqMan analysis, and indicated that MAGE-3 copy numbers equal to or exceeding 1,000 per 104 copies of ß-actin were detectable by conventional RT-PCR techniques. Additional RT-PCR studies indicated that MAGE-3 expression persisted in these cells after cessation of drug exposure. As shown in Figure 1, MAGE-3 mRNA could readily be detected in TE-1 cells 7 days after withdrawal of DAC; MAGE-3 expression in cells treated with sequential DAC/DP could not be evaluated at this time because of the toxicity of this regimen.



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  		Fig 1. Semiquantitative RT-PCR analysis of MAGE-3 expression in the squamous cell esophageal cancer lines TE-1 and TE-13 after exposure to NM, DAC, DP, or sequential DAC/DP. mRNA from the melanoma cell line 586 mel was used as a positive control. Amplification of GAPDH verified the integrity of mRNA.

 
Immunohistochemistry experiments were performed to examine MAGE-3 protein expression in cancer cells after drug manipulation; a representative experiment is depicted in Figure 2. Low-level cytoplasmic expression of MAGE-3 could be detected in TE-1 cells under basal conditions. MAGE-3 expression was clearly enhanced in these cells after DAC exposure, yet only modestly increased after DP exposure. Expression of MAGE-3 in TE-1 cells was most pronounced after sequential DAC/DP exposure; interestingly, this expression was markedly intense yet focal. Although limited quantity of antibody precluded an extensive analysis, similar results were observed in several other cell lines (data not shown).



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  		Fig 2. Immunohistochemistry analysis of MAGE-3 protein expression in TE-1 cells after exposure to NM, DAC, DP, or DAC/DP (A, B, C, and D, respectively) (Magnification x 63).

 
Subsequent experiments were performed to determine whether DAC and DP could inhibit the proliferation of lung and esophageal cancer cells under exposure conditions that facilitated induction of CTA expression. As shown in Figure 3, DAC mediated significant growth inhibition in cancer cells. Depsipeptide also inhibited proliferation of these cells, albeit to a lesser extent than DAC alone. Sequential DAC/DP treatment significantly inhibited the growth of lung and esophageal cancer cells relative to that observed after exposure to normal media or either drug alone. Similar treatment had no significant effects in NHBE cells (data not shown).



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  		Fig 3. Proliferation of H1650 and TE-13 cancer cells after exposure to NM, DAC, DP, or sequential DAC/DP. (*p < 0.001 vs untreated controls; ##p < 0.05 vs cells exposed to DAC or DP alone.)

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Discussion
 References
 
MAGE-3 is one of 12 closely related MAGE genes that are aberrantly expressed in a variety of tumors including lung and esophageal carcinomas [16]. Several recent studies have indicated that MAGE-3 is immunogenic in cancer patients as well as normal individuals [26, 27]. MAGE-3 peptides administered alone or in conjunction with dendritic cells can mediate tumor regression in melanoma patients [6, 7]. However, recognition of MAGE-3 has not been convincingly demonstrated in lung and esophageal cancer patients. Gotoh and associates [28] observed no evidence of specific elimination of cancer cells coexpressing HLA-A2 and MAGE-3 in 22 lung cancer patients, and Stockert and colleagues [29] detected no serologic reactivity against MAGE-3 in 24 lung cancer patients.

Takaki and associates [30] examined binding of MAGE-3 peptides to HLA-A2 in a panel of cultured lung cancer lines. HLA expression was intact in three of five A2 cell lines. All five of the HLA-A2 lines expressed MAGE-3; however, only one line was recognized by CTL specific for MAGE-3. Recognition could be seen against two additional lines after peptide pulsing but not after exposure to IFN-{gamma}, suggesting that the lack of recognition was related to low-level antigen expression rather than to deficiencies regarding antigen processing, which have been well characterized in lung cancer cells—primarily those derived from SCLC [11]. The fact that many lung and esophageal cancers have an intact antigen processing machinery and retain class I HLA expression strongly suggests that low level tumor antigen expression is a major impediment to an efficient host response to these neoplasms.

In the present study, we examined the feasibility of modulating MAGE-3 expression in lung and esophageal cancer cells by 5-Aza-2'-deoxycytidine (deoxyazacytidine) and Depsipeptide FR901228 under exposure conditions potentially achievable in clinical settings. After conversion to a nucleotide, 5-Aza-2'-deoxycytidine, which cannot be methylated, is incorporated into DNA in place of cytosine; this agent also directly inhibits DNA methyltransferase, an enzyme that methylates cytosine residues [31]. Through these two mechanisms, DAC facilitates promoter hypomethylation and induction of a variety of genes including cancer testis and histocompatibility antigens, as well as tumor suppressors [16, 18, 32]. Depsipeptide FR901228 (DP) has recently undergone phase I evaluation at the National Cancer Institute. This novel HDAC inhibitor exhibits cytotoxic activity against a variety of human cancer lines and tumor xenografts; the cytotoxic effects of depsipeptide are less pronounced in cultured normal cells [33, 34]. The mechanisms by which depsipeptide induces growth arrest and apoptosis in cancer cells have yet to be elucidated. We chose to use DAC and DP for our studies because the toxicities and pharmacokinetics of these experimental antineoplastic and differentiation agents have been defined [35].

Our studies demonstrated that under exposure conditions potentially achievable in clinical settings, DAC could induce MAGE-3 expression; DP exhibited very modest activity regarding target gene induction. Sequential DAC/DP treatment mediated pronounced induction of MAGE-3 in essentially all cancer lines but not in normal human bronchial epithelial cells. After drug treatment, MAGE-3 protein expression was readily detectable yet focal in cancer cell lines exhibiting varying proliferation rates; interestingly, focal expression of MAGE-3 has also been noted in a variety of cancer specimens [25]. These observations suggest that MAGE-3 expression may vary during cell cycle progression.

A potential limitation of these studies concerns our inability to demonstrate functional significance of MAGE-3 induction because of the lack of a cytolytic T cell clone to document immune recognition of cancer cells after drug manipulation. These issues are particularly relevant given the documented deficiencies regarding antigen processing and presentation in lung cancer cells. Nevertheless, results of several recent studies indicate that DAC/DP mediated induction of MAGE-3 in lung and esophageal cancer cells may be clinically relevant. Quantitative RT-PCR experiments have revealed that the threshold level of expression of gp100 or FGF5 in melanoma or renal carcinoma cells required for recognition by HLA restricted CTLs specific for these antigens is approximately 500 to 1000 copies per 104 copies of ß-actin; the extent of recognition correlates with increasing levels of antigen expression [22, 36]. MAGE-3 expression exceeded 1000 copies per 104 copies of ß-actin in all of the lung and esophageal cancer cell lines after sequential DAC/DP exposure, and protein levels coincided with mRNA copy number in these cells. Furthermore, several of the cancer lines used in this study (including H-2087 and BE-3) express HLA-A*0201; sequential DAC/DP treatment induces NY-ESO-1 expression in these cells, enabling their recognition by A*0201 restricted CTL specific for this cancer testis antigen (Weiser and colleagues, submitted for publication).

In studies that are described in detail elsewhere, we have observed that under conditions that facilitate induction of cancer testis antigen expression, sequential DAC/DP treatment induces profound apoptosis in cancer cells (Weiser and associates, submitted for publication). These observations may be clinically relevant. Recent data indicate that dendritic cells can acquire MAGE-3 from apoptotic cells to induce a cellular immune response [37]. Thus, in addition to directly enhancing MAGE-3 expression in cancer cells, sequential DAC/DP treatment may conceivably augment antitumor immunity through induction of apoptosis and subsequent release of CTA that can be processed by professional antigen presenting cells within the tumor site. Because sequential DAC/DP simultaneously induces expression of several CTA, this regimen may facilitate immune response against tumors in which individual tumor antigens are focally expressed. Although the mechanisms have not been fully defined, sequential DAC/DP treatment represents a potentially novel strategy to enhance antitumor immunity in patients with thoracic malignancies.


   Discussion
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Discussion
References
 
DR MARK I. BLOCK (San Francisco, CA): I really enjoyed your paper. You present some very interesting results, and I would like to break them down into two findings. First, you demonstrate enhanced expression of antigens and second, you demonstrate growth inhibition and apoptosis. Your fourth conclusion states that you enhance or augment immunogenicity, and I just wanted to probe that conclusion a little bit. Your data show enhanced MAGE-3 expression, but how do you reach the conclusion that this leads to enhanced immunogenicity? Do you have any in vivo data that simply expressing more antigen on the target cell makes it more immunogenic? I’m wondering if immunogenicity is determined not so much by the amount of antigen expressed by the target cell, but rather by the ability of antigen-presenting cells to appropriately activate T-cells that recognize the specific antigen.

DR WEISER: We acknowledge that a limitation of this work is our inability to demonstrate T-cell recognition of the MAGE-3 cancer testis antigen following induction by sequential DAC/DP treatment. We have, however, observed induction and immune recognition of another cancer testis antigen, NY-ESO-1, after sequential drug exposure in lung, esophageal, and mesothelioma cell lines. These data have been submitted for publication and will be presented, in part, at the annual AATS meeting this spring.

DR MALCOLM M. DECAMP, JR (Cleveland, OH): I have a question for you. Did the sequence of DAC and DP exposure affect outcome and, along those lines, could you coexpose the tumor cells to these agents, or is sequence an important step?

DR WEISER: Different treatment regimens were evaluated in our initial experiments with these agents. This presentation highlighted our results after optimization of drug exposure. The timing of drug delivery appears to be crucial, as significantly less gene induction was observed in experiments in which cells were exposed to Depsipeptide before DAC treatment. Preliminary data suggested that simultaneous drug exposure would be too toxic to allow quantitative analysis of gene expression.

DR DECAMP: Does that in turn lead you to any mechanistic speculations about how increased antigen expression can lead to decreased proliferation or increased apoptosis?

DR WEISER: Although no function has yet to be attributed to the cancer testis antigens, we do not believe that MAGE-3 contributes to the growth inhibition and apoptosis observed after exposure to DAC and DP. These agents have global effects on gene regulation and we are currently using cDNA microarray techniques to identify genes that are preferentially expressed after drug exposure. Transfection experiments suggest that MAGE-3 is not toxic to cultured cell lines, and we have observed that an adenoviral construct expressing NY-ESO-1 has no growth inhibitory effects in cancer cells. Thus, we believe that genes other than those encoding cancer testis antigens mediate apoptosis after DAC/DP exposure in cancer cells.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Discussion
 References
 

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