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Ann Thorac Surg 1995;60:1583-1591
© 1995 The Society of Thoracic Surgeons
Division of Cardiovascular and Thoracic Surgery, University of South Florida, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, and Eppley Institute for Cancer Research and Department of Pathology, University of Nebraska Medical Center, Omaha, Nebraska
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Abstract |
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Methods. Established non-small cell lung cancer cell lines A427, SKMES-1, and A549 were grown in the presence of ASODNs complementary to messenger RNA of c-myc, bcl-2, p53, or controls at 1 µmol/L or 10 µmol/L concentrations for 4 or 10 days. Cellular proliferation was measured by tritiated thymidine uptake. Flow cytometry was used to quantitate oncoprotein expression. Intranuclear ASODN uptake was documented by fluoresceine-tagged ASODNs.
Results. Fluoresceine-tagged ASODNs were readily taken up by all cell lines. c-myc, as well as bcl-2 and p53 ASODNs, were found to inhibit proliferation of all cell lines significantly compared with controls, most notably in line A549 (40.1% ± 7.1% of control, p = 0.000 with c-myc ASODN). Antisense c-myc reduced c-myc protein by as much as 71.3% in A427, although protein levels were only minimally reduced in the viable cells of the other lines.
Conclusions. c-myc ASODNs inhibit proliferation of non-small cell lung cancer cell lines as well as reduce c-myc protein expression. Antisense bcl-2 and p53 also cause similar growth inhibition. These results suggest a critical role for activation of these oncogenes in the growth of cultured lung cancer cells. Furthermore, the efficacy and rapid cellular uptake of ASODNs support the potential role of antisense targeting of oncogene expression for pharmacologic control of non-small cell lung cancer.
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Introduction |
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During the past three decades, progress in improving long-term survival with lung cancer treatment has been discouragingly slow. The overall 5-year survival rates for lung cancer have changed little, from 8% in 1960 to 1963 to only 14% in 1983 to 1990, and the absolute number of estimated lung cancer deaths yearly will rise to a record 157,000 in 1995 [1]. In the past 10 years, aggressive multimodality treatment with surgical resection plus cisplatin-based chemotherapy or adjuvant radiotherapy, or a combination of both, has improved the outlook and cure rate slightly for locally advanced cancers [2]. Although progress is certainly being made in controlling local and regional disease, death from distant metastases remains a major hurdle to overcome and new systemic treatment strategies are needed if continued progress is expected.
Advances in the understanding of the molecular genetics of cancer cells have brought about the concept of ``oncogenes'' [3]. These genes differ in structure, expression, or both from their normal counterparts, and somehow appear to be responsible for the malignant transformation of cells. Therefore, oncogenes are obvious targets for the development of novel therapies designed to block their expression. Antisense oligodeoxyribonucleotides (ASODNs) have been widely touted as a promising new class of therapeutic agents for a variety of diseases, including cancer [4, 5]. ASODNs are synthetic analogues designed to bind to the RNA transcripts (messenger RNA or mRNA) of target genes in a sequence-specific manner, thereby interrupting translation of the mRNA into protein and possibly allowing for enzymatic degradation of the original mRNA. The overall result is a specific block of gene expression. The genes selected for targeting by ASODNs as cancer treatment have usually been based on the oncogene/anti-oncogene model of malignancy [4].
Recent attention in cancer research has focused on various genes including c-myc, a protooncogene, whose normal primary effect is related to the stimulation of cell growth and differentiation. Under certain conditions the aberrant expression of c-myc may inappropriately drive the cell through the cell cycle leading to malignant transformation [6–8]. The natural and orderly process of programmed cell death that occurs during the maintenance of tissue homeostasis, so-called apoptosis, appears to be inhibited by another protooncogene bcl-2 that is expressed in high levels in some transformed cells, particularly those resistant to chemotherapy or ionizing radiation [9–12]. Finally, a great deal of attention has been focused on p53 and its role in human cancer. This gene normally functions as a tumor-suppressor gene or anti-oncogene [13], but when mutated it becomes a dominant oncogene expressed in a variety of human malignancies [14], including non-small cell lung cancer [15]. Like bcl-2, p53 and c-myc have also been implicated in the regulation of apoptosis [11]. Indeed, p53 is known to be a modulator of bcl-2 expression.
On the basis of our current understanding of the role these oncogenes play in cancer pathogenesis, suppressing the expression of any of these three might be expected to inhibit the proliferation or viability of malignant cells. Therefore, this study was undertaken to use specific ASODNs created to block expression of these oncoproteins in established squamous cell and adenocarcinoma cell lines to see if growth inhibition would result.
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Material and Methods |
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Synthesis of Oligodeoxyribonucleotides
p53 and bcl-2 phosphorothioate oligodeoxyribonucleotide sequences were designed with help of the OLIGO software program (National Biosciences, Plymouth, MN) to assure negligible self-complementarity. Selected sequences were then compared with those in GenBank to avoid reactivity with known sequences of other genes. c-myc ASODN sequences were selected from previously published results [7, 8]. Table 1
lists the ASODN sequences tested in this study.
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Monitoring Cell Proliferation
Tritiated thymidine incorporation, a proliferation-related assay, was chosen to monitor ASODN effects because the proliferating cell population was expected to be the most sensitive to the ASODNs chosen. Dead and dying cells do not incorporate thymidine into their DNA, so only growing and dividing cells were counted as positive with this assay. Cells were removed from culture flasks using 0.05% trypsin and 0.53 mmol/L EDTA diluted with media and 10% fetal bovine serum. Titers of viable cells were determined by counting trypan blue dye-excluding cells in a hemocytometer.
For cell lines A427 and SKMES-1, cells were seeded in 96-well plates at 1,000 cells/well. For cell line A549, 96-well plates were seeded at a lower density (500 cells/well) because of the more rapid growth of this cell line. Cells were grown for 2 days in media only. After 2 days, fresh media and test 15-mer and 20-mer ASODNs (Table 1
) at 1.0 µmol/L or 10.0 µmol/L concentrations were added to triplicate cultures of each. At day 7, fresh media and ASODNs were added to all wells. At day 9, all cells were given an overnight pulse of tritiated thymidine (Amersham, Arlington Heights, IL) at 25 µL/well from a stock solution of 50 µL tritiated thymidine/mL of media for a final concentration of 3 µCi/mL. On day 10, radiolabeled cells were harvested (Cambridge PHD Cell Harvester; Cambridge Technologies, Watertown, MA) for counting of tritium uptake into proliferating cells. To determine whether fewer days of ASODN exposure would make a difference in the efficacy of ASODNs in inhibiting cell proliferation, one cell line (SKMES-1) was also evaluated after only 4 days of ASODN exposure.
Uptake of ASODN
The fluoresceine-labeled 20-mer phosphorothioate oligodeoxyribonucleotide, which was used to document cellular uptake of the ASODNs, had a random nucleotide sequence by mixing all four nucleotides together during synthesisAu: OK?. Therefore, each ASODN molecule synthesized has a different random nucleotide sequence and, when fluoresceine-labeled, was designated as FAM-Poly N. All three cell lines were tested for FAM-Poly N ASODN uptake. As described in the previous section, cells were incubated with 10 µmol/L FAM-Poly N ASODN for 24 hours. Using procedures described by Jacob and associates [16], cells were then removed from culture flasks using 0.05% trypsin containing 0.53 mmol/L EDTA, then washed in PBS containing 1% bovine serum albumin. Cells (0.5 x 106) were then washed three times with PBS containing 0.3% saponin (Sigma) to lyse cell membranes. A final wash with PBS and bovine serum albumin was followed by fixing in PBS containing 0.5% paraformaldehyde. Samples were then analyzed on an EPICS C Flow Cytometer (Coulter Immunology, Hialeah, FL) at 488 nm with a 5-W argon ion laser, counting 10,000 cell events (nuclei) for percent positivity and mean channel fluorescence (expressed on a log scale) in comparison to negative control tubes. Control tubes consisting of untreated cells for a given experiment were used to determine background log mean channel fluorescence intensities for each set of samples.
Analysis of c-myc, bcl-2, and p53 Protein Expression
Staining for flow cytometric analysis of intracellular protein content of c-myc, bcl-2, and p53 was carried out using modifications of the procedures described by Jacob and associates [16]. After washing in PBS and bovine serum albumin, cell aliquots of 0.5 x 106 cells were first fixed gently by incubating for 10 minutes in 0.5% paraformaldehyde in PBS before staining, because these adherent lung carcinoma cell lines tend to be quite fragile after trypsin/EDTA removal from the culture flasks. Cells were then washed with 1 mL of PBS containing 0.3% saponin to make the cell membrane permeable. Cells were then resuspended in 0.2 mL of PBS containing anti-bcl-2 (Dako, Carpinteria, CA) or anti-c-myc (Oncogene Science, Cambridge, MA) monoclonal antibodies, or an anti-p53 monoclonal antibody cocktail (p53Ab-1 plus p53Ab-2; Oncogene Science) at 1 to 2 µg per aliquot. A second aliquot of cells was stained simultaneously with a isotype-matched monoclonal myeloma control protein (background control) used at the same final concentration as the test antibody. The cells were then incubated with the primary antibodies for 30 minutes at 4°C. After washing three times with PBS, cells were then stained with a fluorescent secondary antibody (FITC goat anti-mouse Ig; Dako) for 30 minutes at 4°C. A final wash in PBS/bovine serum albumin followed. Finally, cells were fixed in PBS containing 0.5% paraformaldehyde.
Samples were then analyzed by flow cytometry, as described above. Percent positivity and log mean channel fluorescence were calculated after subtraction of background fluorescence using EPICS software (Coulter). Background staining differed significantly when comparing one cell line to another, but replicate background for a given cell line was reproducibly similar.
Statistical Analysis
For cell proliferation studies, the mean counts of ASODN-treated cells were compared with counts of control cells and were expressed as the percentage of control counts ± standard deviation in the table and standard errors of the mean in the figures. Statistical analysis for parametric data was performed using the Student's t test in a computer program (Primer of Biostatistics, McGraw-Hill, New York, NY). Differences were considered significant when the p value was less than 0.05.
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Results |
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displays the flow cytometry analysis histograms for nuclear uptake of FAM-Poly N by cell lines A427 and SKMES-1; the histogram for A549 (not shown) was very similar. The intensity of fluorescence shown on the horizontal axis in log scale is directly proportional to the nuclear content of FAM-Poly N in the cell. The peak on the left in each panel represents background control fluorescence of nuclei of cells not incubated with FAM-Poly N. The broader peaks on the right represent intense intranuclear accumulation of FAM-Poly N. The number of nuclei counted for each channel of fluorescence is shown along the vertical axis. The fluoresceine-tagged phosphorothioate oligodeoxyribonucleotide tested, therefore, showed good nuclear uptake after only 24 hours of exposure.
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; Fig 2). For SKMES-1 cells after 4 days exposure, there was a marked reduction in proliferation with ASODNs to three variants of c-myc as well as p53 and bcl-2. Interestingly, with 10 days of ASODN exposure, SKMES-1 cells were somewhat less responsive to antisense c-myc and bcl-2, although antisense p53 was even more effective (Table 2; Fig 3). Also of note, antisense c-myc Control 1, the reverse sequence of antisense c-myc 1, demonstrated a marked inhibitory effect on SKMES-1 cell proliferation at 10 days. This ASODN sequence is not complementary to any part of the c-myc mRNA and presumably achieves its effect through a different mechanism. Finally, A549 cells were the most sensitive to antisense c-myc (all variants) and bcl-2 (Fig 4).
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. This analysis histogram for A427 cells graphically displays the three broad protein peaks corresponding to the three oncoproteins of c-myc, bcl-2, and p53, in contrast to the isotype control antibody peak to the far left on the histogram. The large number of positive cells and high fluorescence intensity indicate substantial expression of these proteins in A427 cells. The results for protein expression in SKMES-1 and A549 cells (not shown) were very similar to that shown in Figure 5.
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. With this flow cytometry technique, the mean channel fluorescence shown on a log scale, displayed on the vertical axis, provides an excellent although relatively quantitative measure of c-myc protein content for comparison among samples of test cells.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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Generally, the antisense agents are synthesized as oligodeoxyribonucleotides rather than their ``ribonucleotide'' analogues because their chemical synthesis is easier, less expensive, and they are relatively more resistant to nuclease degradation [4]. In addition, the backbone holding the nucleotides has been modified chemically from the native phosphodiester linkage to a more nuclease-resistant phosphorothioate (replaces one nonbridging oxygen in each internucleotide phosphate linkage with a sulfur) [5, 7]. To recognize a single mRNA sequence of one particular gene within the entire mRNA population in a human cell, it is believed that the minimum nucleotide length should be 11 to 15 nucleotides, and most successful ASODNs are 15 to 25 nucleotides long [5]. For these reasons, in the present study ASODN backbones consisted of phosphorothioates exclusively or combined with phosphodiester linkages, and 15- (15-mer) or 20 (20-mer)-nucleotide sequences were used. The most common mechanism of action of these agents appears to be the formation of a DNA-RNA hybrid with the target mRNA, which inhibits further ribosomal translation of the mRNA. This may activate an endogenous ribonuclease (RNase H) that can cleave the mRNA at the DNA-binding site releasing the intact ASODN to anneal to another mRNA [4]. The overall effect is to block a specific gene expression, thereby changing the cell phenotype.
Cellular uptake of these charged, high molecular weight ASODNs would be expected to be slow, if at all, into the cell. Nevertheless, studies from a number of laboratories [4, 8], including the present study, using tagged or labeled oligonucleotides, indicate that phosphorothioate ASODNs readily enter the cell cytoplasm and nucleus (Fig 1). Typically, these charged phosphorothioate ASODNs enter the cell by endocytosis [4].
One of the most attractive applications of the antisense strategy has been to modulate oncogene expression. Based on Nobel Prize-winning research on animal RNA tumor viruses in the mid-1970s by Varmus and Bishop [18], specific genes were identified that could transform a cell from a normal to a malignant state, and the genes were termed ``oncogenes.'' Subsequently, normal cellular DNA has been found to contain numerous ``protooncogenes'' that play an irreplaceable role in the growth of normal cells, but contain a latent carcinogenic potential to become an oncogene when exposed to an appropriate mutagenic stimulus. Of the approximate 50,000 to 100,000 genes in the genome, it is estimated that at least 50 can serve as oncogenes and just about 20 have actually been found in mutant form in human tumors [19].
One of the protooncogenes receiving a great deal of attention is in the myc family, which itself was originally discovered in the avian myelocytomatosis virus. The c-myc protooncogene appears to be a nuclear transcriptional regulator that is overexpressed in various tumors including breast cancer and promyelocytic leukemia. Investigators working with both tumors have found cancer cell growth is inhibited when exposed to ASODNs directed against c-myc [7, 8]. As summarized by Edgington [11], c-myc may promote either of two pathways to the resting cell depending on the signal received by the gene: (1) pass through the cell cycle and proliferate; or (2) proceed to cell death by apoptosis. Mutant c-myc in tumor cells may favor the first pathway, particularly when enhanced by expression of another oncogene such as bcl-2 or mutant p53. As a clinical example, patients with Li-Fraumeni syndrome have a high risk for developing malignant tumors and have been found to have germline mutations in p53, in addition to overexpression of c-myc, in their normal fibroblasts [10].
In the present study, c-myc protein was readily expressed in all non-small cell lung cancer cell lines examined (Fig 5). In addition, all of the c-myc ASODN variants tested produced some effect with moderate to severe inhibition of tumor cell proliferation, especially with the capped S variant (see Table 1) after 4 days exposure in the squamous cell carcinoma SKMES-1 line. This ASODN was designed based on the finding that the greatest antisense efficacy was obtained when the target was the 5'-cap region of the mRNA of c-myc [5]. Despite its antiproliferation effectiveness, only a modest decrease in c-myc protein was demonstrated in one cell line (A427) and that cell line had only a moderate inhibition of proliferation. The method of protein assay used in this study may account for this apparent discrepancy, as the flow cytometry antiprotein antibody staining technique only counts the viable cells remaining after ASODN treatment. By their presence, these viable cells indicate that they were resistant in some manner to the anti-c-myc ASODN and might not demonstrate a substantial decrease in the oncoprotein.
Another very different protooncogene garnering considerable interest is bcl-2, initially discovered at the site of translocation on human chromosome 18 in follicular B-cell lymphomas. This gene appears to be the mammalian analogue of the ced-9 gene (cell death gene) found in the tiny Caenorhabditis elegans nematode worm, which is commonly used for basic genetics research [11]. bcl-2 appears to have the ability to block programmed cell death or apoptosis. bcl-2 encodes for a membrane-associated protein localized to the endoplasmic reticulum and the nuclear and outer mitochondrial membranes, and is also widely expressed in embryonic development. This protein does not appear to cause malignant transformation on its own but rather cooperates with other oncogenes such as c-myc to inhibit the apoptosis pathway and induce tumors [20]. Deregulated bcl-2 expression also protects cells from the cytocidal effects of irradiation [9] and anticancer drugs [12], probably by acting as an antioxidant to prevent oxidative damage known to be induced in this setting. In the current preliminary study, ASODNs directed against bcl-2 were especially effective in inhibiting proliferation of the squamous cell carcinoma line SKMES-1 and the adenocarcinoma line A549. The other adenocarcinoma line A427 was relatively insensitive to this ASODN.
Enhanced oncogene expression is only part of the picture emerging in carcinogenesis research. A number of ``tumor suppressor'' genes have also been described recently that appear to function by balancing the strong, growth-stimulating signals released by protooncogenes or their unregulated oncogene counterpart. These growth-suppressing genes may act by participating in apoptosis regulation. The most studied tumor suppressor gene is p53, whose loss or mutation is the most common single lesion in human neoplasia, found in more than one-half of all tumors examined, including lung cancer [15, 21]. A mutated version of p53 acts much like bcl-2 in blocking apoptosis in the face of c-myc expression, extending the life span of the tumor cells [20]. However, mutant p53 protein may even promote cellular proliferation. Some mutant p53 types help transform normal cells into malignant cells 3 to 10 times more efficiently than other oncogenes [22]. Therefore, two possible therapeutic strategies based on p53 have emerged: (1) reintroduce wild-type p53 (normal, unmutated p53) into the cell to protect against malignancy by triggering apoptosis in damaged cells with a neoplastic risk [21], or (2) block mutant p53 expression with specific ASODNs to decrease cell transformation, as well as to allow a wild-type p53 allele possibly still present in the cell to be expressed [19]. In the present study, ASODNs were administered exogenously to block mutant p53 expression, resulting in a marked inhibition of proliferation of the SKMES-1 cells down to just 32% of media-only control growth, with a more modest reduction of growth in the adenocarcinoma cells. Considering the significantly higher incidence of mutated p53 protein expressed in squamous cell carcinoma recently documented in resected lung cancer specimens [15], it not surprising that SKMES-1 cells (a squamous cell carcinoma line) in the present study were the most responsive to anti-p53 ASODNs.
The data presented strongly support the conclusion that the ASODNs used in the present study exerted their effect by nucleotide sequence-specific hybridization with target mRNA, that is, an antisense effect has occurred. Nevertheless, a few findings, such as mild inhibition of cell proliferation with some lines when control c-myc ASODNs were used, also suggest that there may be other mechanisms at work. It is possible that a nonspecific growth inhibition occurred as a result of exposure of the phosphorothioate to the cells, perhaps leading to cell surface receptor binding [5]. Other investigators have documented sequence-independent pleotropic actions of phosphorothioate oligodeoxynucleotides that may involve other cellular targets in addition to specific mRNA transcripts, both selective and nonselective protein binding, and nonselective translation inhibition [23, 24]. Nevertheless, such apparent effects should be minimized in the cell proliferation study by comparing results to various control ASODNs such as Poly I and Control 1. However, even these control ASODNs and the c-myc Control ASODNs demonstrated some effects, especially with SKMES-1.
Another plausible explanation for the inhibitory effects of the ASODNs that may be nonantisense, yet sequence specific, is the recently described phenomenon of aptameric effects [25, 26]. This is the ability of nucleic acids such as DNA oligonucleotides to fold into a stable three-dimensional structure facilitating binding to a specific molecular target (ligand) such as proteins and enzymes. Aptamer effects are sequence specific in that a specific structure will be formed with a specific sequence, but they do not necessarily have a nucleotide sequence that is complementary for antisense effects. This phenomenon is a possible explanation for the inhibitory effects of the ASODNs used in this study.
Another intriguing potential mechanism by which some of the ASODNs used in this study might have caused inhibition of cell proliferation in the lung cancer cell lines relates to a unique structure on eukaryotic chromosomes, the telomere. With each round of replication, chromosomes would tend to lose some terminal bases from the 5' end of each strand of DNA due to the inability of DNA polymerase to completely replicate the chromosome end (telomere), and eventually this continuing DNA loss would be critical leading to cell death [27]. A unique ribonucleoprotein reverse transcriptase called telomerase (also called telomere terminal transferase or DNA-nucleotidylexotransferase) exists in all eukaryotic cells, which adds multiple repeating strands of nucleotides rich in guanines (in human telomeres these sequences are TTAGGG, TTGGGG, or TGAGGG) to the end of chromosomes, always oriented 5' to 3' toward the chromosome's terminus with the 3' end overlapping the 5' end. These telomeres are essential for maintenance of chromosome integrity and stability [28]. The activity of telomerase is essential for protozoans and yeast as well as human germ cells and transformed cells (cancer cell lines such as HeLa cells) that are rapidly growing, but it is not apparently essential for human somatic cells [27].
The RNA in telomerase contains repeating cytosine-rich (C-rich) sequences that are complementary to the telomeric repeats synthesized by the enzyme. It appears that this enzyme is a unique ribonucleoprotein reverse transcriptase whose template for synthesis is actually an intrinsic part of the enzyme. Two studies demonstrating nonsequence-specific inhibitory effects of ASODNs found that the ASODNs with G-rich sequences (those with GGGG sequences) were particularly active [23, 24]; all of the c-myc ASODNs including the partially active c-myc control ASODNs in the present study also have G-rich sequences. Therefore, it is quite possible that these G-rich ASODNs may act by binding the C-rich sequences in telomerase, consequently inactivating the enzyme in these cell lines. Then, the ASODNs would produce their inhibition of proliferation by preventing telomeres from being added to the malignant daughter cell chromosomes at the time of replication, thereby leading eventually to cell death. In fact, it has been suggested that designing drugs to inhibit selectively telomerase might be an effective strategy to treat pathogenic yeasts and protozoans who have significant telomerase activity [27]; perhaps this would also be an effective technique with cancer cells. Of note, telomeres have been described as ``the Achilles heel of chromosomes'' [27].
So what have we learned from this preliminary study with ASODNs and where should we proceed? Currently it appears feasible to design ASODNs complementary to specific mRNAs and expect positive effects to occur in cell culture. This is presumably related to sequence-specific inhibition of mRNA translation although other mechanisms are possible. However, as our understanding of specific gene effects expands, a more precise strategy may emerge that could inhibit more effectively malignant cell proliferation, that will in turn lead to in vivo applications. With the three oncogenes in the present study, the next logical step would be to disrupt the possible cooperation between these oncogenes that enhances tumor growth [10, 20]. For example, ASODNs could be used to inhibit the growth-promoting effects of c-myc and simultaneously add other ASODNs to inhibit the suppression of apoptosis of bcl-2 or to inhibit the enhancement of tumor growth of mutant p53 or its suppression of apoptosis. Alternatively, after ASODN inhibition of the apparent tumor-protective effects of bcl-2, cell death through apoptosis may be more readily achieved with conventional chemotherapy or radiotherapy. Once an effective antisense strategy is found using cell lines in vitro, the next step is to investigate the efficacy of these agents in vivo in treating the lung cancers derived from these same cell lines that can be readily grown in the immunodeficient nude mouse xenotransplant model. Carcinogenesis is undoubtedly a multistep event involving many progressive changes in the cell's genotype and consequently its phenotype [19]. Eventually, ASODNs may make it possible to target abnormal gene expression in this pathway to control and ultimately kill the malignant cells that resulted from these multiple genetic changes.
The step from in vitro cell culture work to intact animal studies is often large and difficult to manage, with test agents often proving too toxic for use in vivo. However, intact animal studies with ASODNs have already begun and have shown a remarkable lack of toxicity with the safe administration of large quantities of these agents to rhesus monkeys [29]. In fact, a preliminary clinical research report has appeared describing the first systemic use of anti-p53 ASODNs administered to humans to treat relapsed or refractory acute myelogenous leukemia [30], thus far documenting the lack of major toxicity of these agents. Such studies pave the way for the future use of ASODNs as a new pharmacologic strategy to modulate gene expression in the treatment of cancer, as well as the potential application of these selective molecular agents to treat a wide variety of acute and chronic diseases that also involve changes in the genome.
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Address reprint requests to Dr Robinson, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Dr, Tampa, FL 33612-9497.
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p < 0.05.)
p = 0.000.)
