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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 602-610 |
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doi:10.1111/j.1745-7270.2006.00205x |
Characterization of Genes
Associated with Different Phenotypes of Human Bladder Cancer Cells
Yu-Cong YANG, Xu LI, and Wei
CHEN*
Center
for Experimental Medicine, the First Affiliated Hospital, School of Medicine, Xi'an
Jiaotong University, Xi'an 710061, China
Received: March 22, 2006�������
Accepted: June 20, 2006
This work was supported by a grant
from the National Natural Science� Foundation of China (No. 30370660)
*Corresponding
Author: Tel, 86-29-85323528; E-mail, [email protected]
Abstract�������� To identify genes associated with
morphological phenotypes of human bladder transitional cell carcinoma, we used
suppression subtractive hybridization (SSH) to create a subtractive cDNA
library of two established cell lines, BLZ-211 and BLS-211, derived from a
patient with transitional cell carcinoma of the bladder, then to screen for
differentially expressed genes. Real-time reverse transcription-polymerase
chain reaction was used to further confirm the selected differentially
expressed genes. Forward and reverse subtractive cDNA libraries yielded 168 and
305 putative clones, and among them more than 90% contained the inserts. After
differential screening, 36 different transcripts were obtained from 64 cDNA
clones of a forward and reverse subtraction library. Among them, 17 were
identified as known genes by homology, for example, Vimentin, Keratin7,
DDH and UCH-L1. The remaining 19 were unknown expressed genes,
and were collected as new expressed sequence tags by the GenBank dbEST database
with the accession numbers DR008207, DR010178, DR159652-DR159660,
DY230447-DY230448, and DY505708-DY505713. Their function will be studied
further. Thus, SSH appears to be a useful technique for identifying
differentially expressed genes between cell lines or clones. Our results, as
revealed by SSH, also suggest that differences in gene expression of
cytoskeletal proteins might contribute to the different morphologies in BLZ-211
and BLS-211 cells.
Key words������� bladder cancer; phenotypes; suppression subtractive
hybridization
Bladder cancer is one of the most commonly diagnosed malignancies in the urinary system.� Eighty percent of these tumors are characterized by transitional cell carcinomas (TCC) with a high recurrence rate (50%-80%) [1]. Intensive� research has been conducted to explore the mechanisms involved in their pathological characteristics. For example, tumor heterogeneity is an important factor that might affect clinical features of the disease [2]. Phenotypic� differences associated with genetic hetero�geneity of the cancer cells might lead to differences in manifested pathology. Thus, analyzing the genetic heterogeneity and gene expression could provide insight for a biological basis of bladder cancer.
We have previously established two different cell lines, named BLZ-211 and BLS-211, originally derived from the surgical sample of a 55-year-old male patient with grade II TCC of the bladder through routine pathological diagnosis� [3]. We have previously demonstrated that these two cell lines were clonally established and appeared to be useful models with respect to the questions of genetic hetero�geneity in bladder cancer [4-6]. In order to exclude the effect of culture in vitro, we detected all cell cultures biological� characteristics within 60 passages. These two cell lines share some similar characteristics. For example, the DNA index is triploidy in both lines and the values, determined by flow cytometry, are 1.602 and 1.591 for BLZ-211 and BLS-211 cell lines, respectively. They contain� at least five shared structural anomalies as revealed by detailed karyotyping [4] and both are sensitive to cisplatin, but not to vincristine, in drug sensitivity testing [5].
However, these two cell lines show remarkable dif�ferences in cell morphology. BLZ-211 cells are polyhedral and plump, with large vesicular nuclei and nucleoli; and the cells grow in a tightly connected manner with virtually no space between them. By comparison, BLS-211 cells are spindle-shaped with elongated and partly twisted characteristics, and do not have a polarized epithelial phenotype� or tight connection between them. BLS-211 cells are also more sensitive in response to trypsin digestion than BLZ-211 cells. In addition, cell adhesion assay revealed� that the amount of BLZ-211 cells adhering to fibronectin is higher than that of BLS-211 cells [6] and in vitro scrape wound assay showed that the speed of BLS-211 migration is faster than that of BLZ-211. Cloning efficiency in soft agar is 12% and 6% for BLS-211 and BLZ-211 cells, respectively. As these two cell lines were originally cloned from the same tumor sample and had similar chromosomal alterations, we speculate that the difference� in gene expression might contribute to these morphological differences. Identification and characteriza�tion of the differences in gene expression between them could provide important clues for the understanding of the pathological basis and the discovery of new molecular targets associated with the pathogenesis of bladder cancer.
A number of different
techniques have been used to characterize the differences in gene expression.
These include� subtractive hybridization [7], differential display [8],
representational difference analysis (RDA) [9], cDNA array hybridization [10]
and serial analysis of gene expression� [11]. The technique of cDNA microarray
has been commonly used to allow the generation of differential� expression
profiles, but it cannot find novel sequences that are not yet available on the
microchips. One recently described� method, suppression subtractive
hybridization (SSH) [12], is similar to RDA but with some extra advantages. For
example, the SSH procedures allow the identification of differentially
expressed genes, particularly for those genes whose levels of expression are
"significantly" different between two target populations without
amplification of commonly expressed genes. SSH can isolate� unknown cDNAs for
the study of gene expression.
In the present work, we used
SSH to explore the dif�ferences in gene expression between BLZ-211 and BLS-211
cell lines, which might determine the genetic components associated with their
morphological variations.
Materials and Methods
Cell storage and culture
The BLZ-211 and BLS-211 cell lines were originally established� in 1993 from a 55-year-old male patient with a grade II TCC of the bladder. The cells were kept in liquid nitrogen. For cell culture, they were maintained in RPMI 1640 supplemented with 2 mM L-glutamine, 10% fetal calf serum, 400 U/ml penicillin, and 200 mg/ml streptomycin, at 37 �C in a 5% CO2 atmosphere.
Scrape wound
migration/proliferation assay
The potential for migration and proliferation of BLS-211 and BLZ-211 cells were assessed using an in vitro scrape wound assay. The two kinds of cells were plated in a 50 ml culture bottle (5105 cells). At confluence, monolayers were scraped using a sterile stick to make a straight groove in the cell monolayer. Cell culture media were aspirated and replaced by 8 ml of fresh RPMI 1640. At 0, 24 and 48 h after wounding, cells were viewed and photographed.
RNA isolation and cDNA library
generation for SSH
Cells were grown to 80%-90% confluence. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, USA) following the protocols provided by the supplier, and then dissolved in nuclease-free water. mRNA was extracted using a Poly(A) pure kit (Qiagen, VIC, Australia). Using 2 mg of mRNA, cDNA synthesis was carried out with a PCR-select cDNA subtraction kit (Clontech, Palo Alto, USA) according to the recommendation from the supplier. SSH of the cDNA was then carried out using the advantage� cDNA polymerase included in the same kit. By reversing the tester and the driver, two subtractive libraries were generated� in order to isolate upregulated transcripts� in both BLZ-211 and BLS-211 cells. The SSH-cDNA repertoire for each experiment was cloned into the pGEM-T easy vector (Promega, Madison, USA) and transformed� into Escherichia� coli JM109, then stored at -80 �C.
Differential screening using
subtracted probes
Colonies from each library were selected and the inserts� amplified by PCR using adaptor primers (5'-TCGAGCGGCCGCCCGGGCAGGT-3' and 5'-AGCGTGGTCGCGGCCGAGGT-3') and Taq polymerase. The PCR products were analyzed, sized by gel electrophoresis, then arrayed in duplicate by manually spotting onto nylon membranes and fixed at 80 �C in an oven. The arrays were screened by hybridization using 32P-labeled unsubtracted and subtracted� cDNA probes which were generated from BLZ-211 and BLS-211 SSH-cDNA by random priming (Clontech). Prior to labeling, the adaptor sequences were removed from the SSH-cDNA by digestion with RsaI and SmaI, followed by purification using a QIAquick kit (Clontech). Membranes were hybridized with 32P-labeled probes at 42 �C and were detected by autoradiography. Colonies that showed differences 2-fold greater in signal intensity as assessed by densitometry were taken for further� analysis.
DNA sequencing
Sequencing of all cDNA clones in the two SSH libraries was carried out on ABI 377 DNA Sequencer at Shanghai Huanuo Biotechnology Company (Shanghai, China) using M13 forward primer. Nucleic acid sequence homology searches were performed by comparison to the National Center for Biotechnology Information's RefSeq, GenBank, and expressed� sequence tags databases (dbEST) using BLAST (http://www.ncbi.nlm.nih.gov/blast).
Reverse transcription (RT)-PCR
analysis
Total RNA (1 mg) from the cells was reversely transcribed to cDNA by SuperScript II reverse transcriptase (Invitrogen) in a 20 ml reaction volume. PCR was performed� with 0.5 ml cDNA template, 1�reaction buffer, 0.2 mM dNTP mix, 25 mM each primer and 0.3 u Taq DNA polymerase (TaKaRa, Dalian, China), and subjected to 30 cycles of amplification at 94 �C for 30 s, 54 �C for 30 s, and 72 �C for 40 s. The G3PDH was used as an internal control in each PCR amplification. The PCR primers for each individual gene were synthesized by TaKaRa and Shanghai Huanuo Biotechnology Company. The PCR products� were separated by electrophoresis in 1.5% agarose� gel stained with ethidium bromide and photographed under� ultraviolet light.
Real-time RT-PCR analysis
Using real-time RT-PCR, four genes were chosen for further analysis. Primers of target genes were designed and synthesized by TaKaRa, and are listed in Table 1. A 7300 real-time PCR system was used (Applied Biosystems, Foster City, USA). PCR reactions were performed in a total volume of 25 ml containing 12.5 ml 2�SYBR premix ExTag (TaKaRa), 0.5 ml 50�Rox Reference Dye (TaKaRa), 1.0 ml each primer (200 ng) and 1.0 ml cDNA of BLS-211 or BLZ-211 cells in each sample. In order to guarantee the comparability� of the calculated gene mRNA expression in all analyzed samples, the housekeeping gene G3PDH was amplified. The amplification program included a denaturation step (10 s at 95 �C) and an amplification step (5 s at 95 �C, 15 s at 59 �C, 31 s at 72 �C, 40 cycles), followed by a melting curve program.
Results
Morphologic characteristics
under light microscopy
As shown in Fig. 1, BLZ-211 cells appeared to have a polarized epithelial phenotype and grow in a tightly connected� manner [Fig. 1(A)], whereas BLS-211 cells were spindle-shaped and loosely distributed, as previously described [Fig. 1(B)].
Scrape wound
migration/proliferation assay
Fig. 2 shows representative images of the wound scrape after 0, 24 and 48 h for each group of cells. After 24 h, BLS-211 cells had completely healed the wound, but BLZ-211 cells had only healed approximately 60% of it. After 48 h, BLZ-211 had healed 90% of the wound. BLS-211 cells have the ability to migrate and proliferate more quickly than BLZ-211 cells.
cDNA library generation
We applied SSH to mRNAs isolated from BLZ-211 and BLS-211 cell lines. The subtractions were conducted in libraries for both directions (BLZ-211 minus BLS-211) and (BLS-211 minus BLZ-211). Two SSH cDNA libraries were constructed. They were considered to be of good quality in determining the efficiency of the subtraction. We used 2 ml secondary PCR products and 1 ml T-easy vector for ligation, and 50 ml high efficiency competent cells (1�108 cfu/mg DNA) were used for transformations. The subtractive forward and reverse cDNA libraries had 168 and 305 white colonies, respectively. White colonies should contain inserts, whereas blue colonies should not. We found that over 90% of the white colonies contained inserts.
Differential screening
cDNA clones from each library were arrayed in a duplicate manner by spotting onto nylon membranes and screened with reverse hybridization using subtracted and unsubtracted cDNA probes. The screening of the subtracted clones permitted ready identification of the genes that were differentially expressed and substantially increased� the efficiency with which colonies of interest could be identified (Fig. 3). Of the clones analyzed, 64 (37 in BLZ-211 and 27 in BLS-211 cells) were detected as overexpressed in one cell line relative to the other, based on the criteria of a greater than 2-fold difference in hybri�dization� signal intensity.
Identification of
differentially expressed genes
Thirty-six different transcripts were found among the 64 clones isolated. Tables 2 and 3 show the upregulated genes in BLZ-211 and BLS-211 cells, respectively. Among them, 17 were identified as known genes by homology, and 19 were genes of unknown function, as determined by searching the GenBank database. These 19 were unknown transcripts, and were collected as new expressed sequence tags by the GenBank dbEST database with the accession numbers DR008207, DR010178, DR159652-159660, DY230447-230448, and DY505708-DY505713.
RT-PCR analysis
We used RT-PCR to further examine the isolated genes (Fig. 4). With RT-PCR, we found that Vimentin (PCR product� 119 bp) was overexpressed in BLS-211 cells and Keratin7 (PCR product 111 bp) and Fibronectin (PCR product 143 bp) were overexpressed in BLZ-211 cells.
Real-time RT-PCR analysis
mRNA expression profiles of the four candidate genes were analyzed. Following amplification, CT (threshold cycle), DCT (normalization of CT for target gene relative to G3PDH CT) and DDCT (comparing differences in the DCT values of BLS-211 to BLZ-211) values were calculated and are shown in Table 4. Relative quantifications of the expression levels of the four candidate genes between BLS-211 and BLZ-211 were analyzed using real-time RT-PCR. We confirmed that expression levels of Vimentin and TAL6 increased in BLS-211, Fibronectin and Keratin7 decreased in BLS-211 (Table 4).
Discussion
SSH is a powerful approach for the identification of genes that are differentially expressed in one cell population compared with another. Although cDNA microarray is increasingly applied for massive parallel analysis of gene expression, SSH is still widely used as it enables the recovery of abundant, as well as low copy-number, mRNA transcripts. In addition, SSH not only permits efficient identification of phenotype-associated genes with known function, but also allows the unbiased isolation of novel sequences that are not yet available on the microchips. But the most tedious step of this method is validation of the clones.
In this work, we arrayed and screened initial SSH clones using reverse hybridization that could minimize the number of non-relevant clones. We then further verified some genes by real-time RT-PCR. In order to exclude the potential genomic DNA contamination in RT-PCR, we used DNase to isolated RNA, and a negative control (contains RNA and all other reagents except for the reverse transcriptase) during the cDNA synthesis portion was included. Using these tools, we compared two TCC cell lines characterized by different morphology. We found that 17 known genes and 19 unknown genes were differentially expressed. Two cytoskeletal genes, Keratin7 and Vimentin, were identified. Keratin7 was overexpressed in BLZ-211 cells, whereas Vimentin was overexpressed in BLS-211 cells. Both encode intermediate filament proteins. Normal adult urothelium is known to express keratin subtypes with the characteristic of "simple" epithelia (K7, K8, K18, K19 and K20) and "stratified" epithelia (K13). K7 is retained by all bladder TCCs, and is regarded as a bladder epithelial tumor marker [13]. Vimentin is regarded as a mesenchymal marker and is not expressed in normal adult urothelium. But in our study, Vimentin was upregulated in the BLS-211 cell line. The cytoskeletal gene expressions are consistent with phenotypic differences in cell shape, which suggests that they might contribute to the spindle shape for BLS-211 cells and the epithelial shape for BLZ-211 cells. We also detected Vimentin and Keratin7� expression in two cell lines of human cervical carcinomas, CS1213 and RJC. These two cell lines displayed anchor-dependent or anchor-independent characteristics. The anchor-independent cell line (CS1213) displayed elliptic or spindle cell shape and overexpressed the Vimentin gene, but the anchor-dependent cell line (RJC) showed epithelial phenotype and highly expressed the Keratin7 gene, which confirmed that cytoskeletal genes can affect cell shape and growth.
We found that, in in vitro scrape wound assays, healing from high-strength induced wound in BLS-211 cells took place at 24 h, but the process took more than 48 h in BLZ-211 cells. This suggests that the migration of BLS-211 cells is faster than that of BLZ-211 cells. In addition, cloning efficiency in soft agar is higher in BLS-211 (12%) than that in BLZ-211 (6%). This might suggest that BLS-211 cells are relatively more invasive. Furthermore, BLS-211 cells are established from bladder TCC and also overexpress epithelial marker TAL6, a tumor-associated antigen L6, or transmembrane 4 superfamily member 1. TAL6 has been reported to be associated with cell motility and metastatic potential of solid tumor [14,15]. This would suggest that BLS-211 is a type of epithelial-mesenchymal transition (EMT) cells. In vitro, EMT results in increased cell motility, loss of epithelial morphology and acquisition of mesenchymal characteristics such as expression of intermediate filament vimentin [16]. Therefore, the BLS-211 cell line can be a model for studying EMT in bladder TCC.
Mitochondrial genes that overexpressed in BLZ-211 cells included NADH dehydrogenase, ATP synthase 6 and cytochrome c oxidase. These genes are associated with cell energy metabolism. This reflects that BLZ-211 cells are metabolically highly active. These findings are in accordance with previous electron microscopy results showing that BLZ-211 cells have larger and increased numbers of mitochondria [3].
Certain genes encoding enzymes were overexpressed in BLS-211 cells, such as UCHL1 (ubiquitin thiolesterase), TXNRD1 (thioredoxin reductase 1), and DDH (dihydrodiol dehydrogenase 1). They might also be involved in phenotypic differences in BLS-211 morphology. UCHL-1 protein is a thiol protease that hydrolyzes a peptide bond at the C-terminal of ubiquitin and is involved with the processing of ubiquitin precursors and ubiquinated proteins. Studies have suggested that ubiquitin plays an important role in EMT. The ubiquitination of E-cadherin is essential for the shuttling of E-cadherin to the lysosome and its degradation. E-cadherin is a type I transmembrane protein of the adherens junctions and is degraded in EMT [17]. Thioredoxin reductase 1 (TrxR1) is a cytosolic enzyme that plays a central role in controlling cellular redox homeostasis. TrxR1 can transduce regulatory redox signals through NADPH-dependent reduction of thioredoxin (Trx), which is able to reduce a broad spectrum of target enzymes and regulate the activity of several transcription factors (e.g., p53 and NF-kB). The TrxR1/Trx system is involved in every step of cancer biology, ranging from transformation and progression to invasion, metastasis and resistance to therapy [18]. Therefore, they might contribute to the morphological phenotype of BLS-211. Dihydrodiol dehydrogenase is a member of the aldo-keto reductase superfamily, which changes the aldehyde or ketone moiety to a corresponding alcohol by using NADH or NADPH as a cofactor [19]. Overexpression of DDH has been found in some cancers, for example, breast, lung, esophageal, cervical and prostate cancers [20-24], and has been associated with disease progression. Our finding that the overexpression of these genes might be related to the morphological features of BLS-211 appears to be the first time such a relationship has been described in bladder cancers, and needs to be confirmed further.
In summary, these results suggest that phenotypic differences in cell shape could be related to genes regulating the cytoskeleton and enzymes. Differentially expressed genes found in this work provide an important direction for further exploration of human bladder TCC progression.
References
1�� Kurth KH, Bouffioux C, Sylvester R, van der
Meijden AP, Oosterlinck W, Brausi M. Treatment of superficial bladder tumors:
Achievements and needs. The EORTC Genitourinary Group. Eur Urol 2000,
37(Suppl): 1-9
2�� Gildea JJ, Golden WL, Harding MA, Theodorescu
D. Genetic and phenotypic changes associated with the acquisition of
tumorigenicity in human bladder cancer. Gene Chromosome Cancer 2000, 27: 252-263
3�� Li X, Chen W, Nan XY, Yan CF, Yang YC, Cheng
XL. The biological characteristics of a newly established human bladder
transitional cell carcinoma cell line BLZ-211. Chin J Urol 1997, 18: 328-331
4�� Yan CF, Li X, Chen W. Karyotypic
characteristics of three new established human bladder transitional cell
carcinoma lines. Journal of Xi�an Medical University 1997, 18: 427-429
5�� Lin R, Li X, Chen W. Chemosensitivity testing
of three novel bladder transitional cell carcinoma lines derived from an
untreated patient and BIU-87 cell line. Journal of Xi�an Medical University
2000, 21: 39-41
6�� Chen W, Li X, Cheng XL, Lin R. Study on
invasion related factors of three human urinary bladder cancer cell lines.
Journal of Xi�an Jiaotong University (Medical Sciences) 2004, 25: 89-91
7�� Davis MM, Cohen DI, Nielsen EA, Steinmetz M,
Paul WE, Hood L. Cell-type-specific cDNA probes and the murine I region: The
localization and orientation of Ad alpha. Proc Natl Acad Sci USA 1984, 81: 2194-2198
8�� Liu YG, Xiong YZ, Deng CY. Isolation,
sequence analysis and expression profile of a novel swine gene differentially
expressed in the Longissimus dorsi muscle� tissues from Landrace�Large white cross-combination. Acta Biochim
Biophys Sin 2005, 37: 186-191
9�� Lisitsyn N, Lisitsyn N, Wigler M. Cloning the
differences between two complex genomes. Science 1993, 259: 946-951
10� Schena M, Shalon D, Davis RW, Brown PO.
Quantitative monitoring of gene expression patterns with a complementary DNA
microarray. Science 1995, 270: 467-470
11� Velculescu VE, Zhang L, Vogelstein B, Kinzler
KW. Serial analysis of gene expression. Science 1995, 270: 484-487
12� Diatchenko L, Lau YF, Campbell AP, Chenchik A,
Moqadam F, Huang B, Lukyanov S et al. Suppression subtractive
hybridization: A method for generating differentially regulated or
tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 1996, 93:
6025-6030
13� Langner C, Wegscheider BJ, Rehak P, Ratschek
M, Zigeuner R. Prognostic value of keratin subtyping in transitional cell
carcinoma of the upper urinary tract. Virchows Arch 2004, 445: 442-448
14� Hashida H, Takabayashi A, Tokuhara T, Hattori
N, Taki T, Hasegawa H, Satoh S et al. Clinical significance of
transmembrane 4 superfamily in colon cancer. Br J Cancer 2003, 89: 158-167
15� Kao YR, Shih JY, Wen WC, Ko YP, Chen BM, Chan
YL, Chu YW et al. Tumor-associated antigen L6 and the invasion of human
lung cancer cells. Clin Cancer Res 2003, 9: 2807-2816
16� Arias AM. Epithelial mesenchymal interactions
in cancer and development. Cell 2001, 105: 425-431
17� Palacios F, Tushir JS, Fujita Y,
D�Souza-Schorey C. Lysosomal targeting of E-cadherin: A unique mechanism for
the down-regulation of cell-cell adhesion during epithelial to mesenchymal
transitions. Mol Cell Biol 2005, 25: 389-402
18� Gorreta F, Runfola TP, Vanmeter AJ, Barzaghi
D, Chandhoke V, Del Giacco L. Identification of thioredoxin reductase
1-regulated genes using small interference RNA and cDNA microarray. Cancer Biol
Ther 2005, 4: 1079-1088
19� Vogel K, Bentley P, Platt KL, Oesch F. Rat
liver cytoplasmic dihydrodiol dehydrogenase. Purification to apparent
homogeneity and properties. J Biol Chem 1980, 255: 9621-9625
20� Ji Q, Aoyama C, Nien YD, Liu PI, Chen PK,
Chang L, Stanczyk FZ et al. Selective loss of AKR1C1 and AKR1C2 in
breast cancer and their potential effect on progesterone signaling. Cancer Res
2004, 64: 7610-7617
21� Hsu NY, Ho HC, Chow KC, Lin TY, Shih CS, Wang
LS, Tsai CM. Overexpression of dihydrodiol dehydrogenase as a prognostic marker
of non-small cell lung cancer. Cancer Res 2001, 61: 2727-2731
22� Wang LS, Chow KC, Wu YC, Lin TY, Li WY.
Inverse expression of dihydrodiol dehydrogenase and glutathione-S-transferase
in patients with esophageal squamous cell carcinoma. Int J Cancer 2004, 111:
246-251
23� Ueda M, Hung YC, Chen JT, Chiou SH, Huang HH,
Lin TY, Terai Y et al. Infection of human papillomavirus and
overexpression of dihydrodiol dehydrogenase in uterine cervical cancer. Gynecol
Oncol 2006, 102: 173-181
24� Stanbrough M, Bubley GJ, Ross K, Golub TR,
Rubin MA, Penning TM, Febbo PG et al. Increased expression of genes
converting adrenal androgens to testosterone in androgen-independent prostate
cancer. Cancer Res 2006, 66: 2815-2825