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Tumor Biology |
Department of Radiation Oncology, Long Island Jewish Medical Center, The Long Island Campus for the Albert Einstein College of Medicine, New Hyde Park, New York 11040 [R-q. Y., S. F., I. D. G., E. M. R.]; Departments of Radiation Oncology [M. A., E. M. R.] and Developmental and Molecular Biology [E. M. R.], Albert Einstein College of Medicine, Bronx, New York 10461; and Metabolism Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892 [D. M. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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phosphatidylinositol-3- kinase c-Akt. However, the downstream alterations in gene expression resulting from this pathway have not been established. On the basis of cDNA microarray and semiquantitative RT-PCR assays, we found that MDA-MB-453 human breast cancer cells preincubated with HGF/SF and then exposed to Adriamycin (ADR), a DNA topoisomerase II inhibitor, exhibit an altered pattern of gene expression, as compared with cells treated with ADR only. [HGF/SF+ADR]-treated cells showed altered expression of genes involved in the DNA damage response, cell cycle regulation, signal transduction, metabolism, and development. Some of these alterations suggest mechanisms by which HGF/SF may exert its protective activity, e.g., up-regulation of polycystic kidney disease-1 (a survival-promoting component of cadherin-catenin complexes), down-regulation of 51C (an inositol polyphosphate-5-phosphatase), and down-regulation of TOPBP1 (a topoisomerase IIB binding protein). We showed that enforced expression of the cdc42-interacting protein CIP4, a cytoskeleton-associated protein for which expression was decreased in [HGF/SF+ADR]-treated cells, inhibited HGF/SF-mediated protection against ADR. The cDNA microarray approach may open up new avenues for investigation of the DNA damage response and its regulation by HGF/SF. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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inhibitor that induces single- and double-strand DNA breaks similar to those induced by ionizing radiation. Interestingly, preincubation with HGF/SF also reduced the number of residual DNA strand breaks at 24 h after exposure to ADR or ionizing radiation, suggesting that HGF/SF may also enhance the rate of DNA repair (i.e., strand rejoining; Ref. 6 ). The increased DNA repair and the cell protection against DNA damage appeared to be attributable to at least in part, to: (a) activation of a cell survival pathway involving PI3K and c-Akt (protein kinase B); and (b) subsequent stabilization of the protein levels of the antiapoptotic mitochondrial pore-forming protein Bcl-XL (5 , 6) .
These studies have not revealed the downstream effector genes that mediate cytoprotection by HGF/SF. Cytoprotection by HGF/SF might involve nonnuclear events, such as inactivation of proapoptotic effectors (e.g., Bad and caspase-9) by c-Akt-mediated protein phosphorylation events (8 , 9) . However, it might also involve prolonged patterns of altered gene expression induced by HGF/SF in the DNA-damaged cells. The latter possibility was suggested by the observation that maximal protection required a preincubation of cells with HGF/SF for at least 48 h before exposure to ADR (5) . Shorter preincubation periods yielded less protection, and application of HGF/SF only at the time of ADR treatment and during the 72-h postincubation period gave no protection.
To investigate the potential alterations of gene expression that might contribute to HGF/SF-mediated cell protection, we have used a cDNA microassay approach, using a previously studied model for HGF/SF protection (5) . MDA-MB-453 human breast cancer cells were preincubated with HGF/SF, exposed to ADR, and then postincubated in ADR-free culture medium for 72 h to allow the repair processes to proceed. Alterations of mRNA expression were examined in cells treated with [HGF/SF+ADR], in comparison with cells treated with ADR alone.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines and Culture.
MDA-MB-453 human breast cancer cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in DMEM supplemented with FCS (5% v/v), nonessential amino acids (100 mM), L-glutamine (5 mM), streptomycin (100 µg/ml), and penicillin (100 units/ml; all from BioWhittaker, Walkersville, MD). Cells were grown at 37°C in a humidified atmosphere of 95% air and 5% CO2.
ADR Treatment.
Subconfluent proliferating cells in 100-mm plastic dishes or 96-well plates were preincubated in the absence or presence of HGF/SF (100 ng/ml x 48 h) in serum-free DMEM and then sham-treated (control) or treated with ADR (10 µM x 2 h, at 37°C) in complete culture medium (DMEM plus 5% FCS). Cultures were then washed three times to remove the ADR and postincubated in fresh drug-free complete culture medium at 37°C for 72 h (again in the absence or presence of HGF/SF, respectively). Cultures were then harvested for isolation of total cell RNA and cDNA microarray or semiquantitative RT-PCR analyses.
Transient Transfections.
Subconfluent proliferating cells were transfected overnight using Lipofectamine (Life Technologies, Inc., Rockville, MD; 10 µg of plasmid DNA/100-mm dish) and then washed to remove the excess vector and Lipofectamine. As a control for transfection efficiency, cultures were cotransfected with 10 µg of a ß-galactosidase expression vector (pSV-ß-gal; Promega Corp., Madison, WI) under parallel conditions; and ß-galactosidase was detected using a 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining kit (Gene Therapy Systems, Inc., San Diego, CA).
MTT Cell Viability Assay.
This assay is based on the ability of viable mitochondria to convert MTT, a soluble tetrazolium salt into an insoluble formazan precipitate, which is dissolved in dimethyl sulfoxide and quantitated by spectrophotometry (11)
. To test the effect of CIP4 on HGF/SF-mediated cell protection, cells transiently transfected with CIP4 expression vectors (see above) were harvested using trypsin and seeded into 96-well dishes (2000 cell/well) in standard growth medium, incubated for 24–48 h to allow attachment and entry into the cell cycle, preincubated ± HGF/SF (100 ng/ml x 48 h), treated with ADR (10 or 20 µM x 2 h), postincubated for 72 h, and tested for MTT dye conversion. Cell viability was calculated as the amount of MTT dye conversion relative to sham-treated control cells. Ten replicate wells were tested for each experimental condition. Statistical comparisons were made using the two-tailed Student’s t test.
Isolation of RNA.
After cell treatments ± ADR ± HGF/SF, the total cellular RNA was extracted using TRIzol Reagent (Life Technologies, Inc.), according to the manufacturer’s instructions. The RNA was treated with DNase and precipitated using 95% ethanol prior to cDNA synthesis. Isolated RNA was electrophoresed through 1.0% agarose-formaldehyde gels to verify the quality of the RNA, and RNA concentrations were determined from absorbance measurements at 260 and 280 nm.
cDNA Synthesis and Microarray Hybridization.
One hundred µg of total cellular RNA was annealed to oligo(dT) and reverse-transcribed in the presence of Cy3-labeled or of Cy5-labeled dUTP (Amersham Pharmacia Biotech, Piscataway, NJ), using 10,000 units/ml of Superscript II reverse transcriptase (Life Technologies, Inc.). The resulting Cy3- and Cy5-labeled cDNAs were treated with RNase One (Promega) for 10 min at 37°C, combined, purified by using a Centricon-50 filtration spin column (Millipore, Bedford, MA), and concentrated to a final volume of 6.5 µl. The cDNA was then combined with 12.5 µl of hybridization solution and 1.0 µl of blocking solution to a final volume of 20 µl. The mixture was heated at 94°C for 2 min and centrifuged at 13,000 rpm for 10 min, and the supernatant was transferred to a clean tube and incubated at 50°C for 1 h.
Hybridizations were performed on cDNA microarray glass slides prepared at the Albert Einstein College of Medicine microarray facility. Each slide contained 9216 unique human cDNA clones. The hybridization solution was placed on a pretreated microarray slide, covered with Hybri-slip, and then incubated in a hybridization chamber overnight at 50°C. After hybridization, the slide was washed at room temperature, first with 0.2 x SSC, 0.1% SDS for 20 min with gently shaking, and then with 0.2 x SSC two times (20 min each time). The slide was dried by spinning at low speed in a centrifuge for 5 min.
Scanning, Griding, and Analysis.
The slides were scanned using a Microarray Scanner 4000A (Axon Instruments) at the Albert Einstein College of Medicine Cancer Center microarray facility. The scanner output images were localized by overlaying a grid on the fluorescent images, using the ScanAlyze software by Michael Eisen, Stanford University.4
The fluorescent intensities were then calculated, using the program Copy of FUBAR! (the easy way out). The final reported intensity was the difference between average probe intensity and average local background intensity. Both final reported intensities (green and red) were filtered, and the spots with intensity <1.5 were eliminated. The ratios of the red intensity to the green intensity and green intensity to red intensity for all targets were determined. The cDNA microarray results comparing cells treated with (HGF/SF+ADR) versus ADR alone are based on three completely independent experiments involving separate cell treatments, separate RNA isolations, and separate microarray assays. The microarray results comparing cells treated with HGF/SF alone vs 0 (control) are based on two completely independent experiments.
Semiquantitative RT-PCR Analysis.
Aliquots of total cellular RNA (1.0 µg) were subjected to first-strand cDNA synthesis using Superscript II reverse transcriptase (Life Technologies, Inc.), and the cDNA was diluted five times with water. One µl of the diluted cDNA was used for each PCR reaction. PCR amplifications were performed using a Perkin-Elmer DNA thermal cycler. The PCR primer sets used in this study are shown in Table 1
. The PCR reaction conditions were individually optimized for each gene product studied. For each gene product, the cycle number was adjusted so that the reactions fell within the linear range of product amplification. PCR reaction conditions and cycle numbers are shown in Table 2
. The ß-actin and ß2-microglobulin genes were used as controls for loading. PCR products were analyzed by electrophoresis through 1.2% agarose gels containing 0.1 mg/ml of ethidium bromide, and the gels were photographed under ultraviolet illumination. The amplified cDNA product bands were quantitated by densitometry.
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Western blotting was performed as described earlier (5) . The immunoprecipitated proteins or equal aliquots of total cell protein (50 µg/lane) were electrophoresed, transferred, and blotted using the appropriate primary antibody. The primary antibodies were: (a) anti-c-Met antibody H-190 (sc-8307, rabbit polyclonal IgG; Santa Cruz Biotechnology; 1:500 dilution); (b) anti-phosphotyrosine antibody (Ab-4, mouse monoclonal; Calbiochem/Oncogene Research Products; 1:500 dilution); and (c) an anti-myc mouse monoclonal antibody (Invitrogen, Carlsbad, CA) at a 1:1500 dilution, to detect the myc epitope tagged wild-type and mutant CIP4 proteins.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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inhibitor that induces single- and double-stranded DNA breakage. The basic experimental protocol is described in "Materials and Methods" and is summarized in the diagram shown below:
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This design was chosen for several reasons. The main comparison was between [HGF/SF+ADR] versus ADR alone to identify genes for which expression was altered by HGF/SF during the response to DNA damage, because it is likely that some of these alterations may contribute to HGF/SF-mediated cell protection. However, a comparison of cells treated with HGF/SF versus CONTROL (sham treatment only) was also made. A postincubation period of T = 72 h after removal of ADR was used to examine well-established alterations in gene expression rather than transient changes occurring immediately after DNA damage. Furthermore, alterations in mRNA levels observed at T = 72 h are more likely to reflect changes in protein protein levels, because the mRNA alterations are of a prolonged duration.
Previous studies indicate that the ability of HGF/SF to protect cells against DNA-damaging agents is attributable to a c-Met receptor-mediated signaling pathway leading to the activation of a c-Akt-dependent survival pathway (6
, 7)
. The HGF/SF-mediated cell protection was blocked by two fragments of the HGF/SF protein (designated NK1 and NK2) that bind strongly to the c-Met receptor, fail to fully activate c-Met signal transduction, and function as competitive antagonists of the full-length HGF/SF protein (5)
. Here, we show by IP-Western blotting that exposure of MDA-MB-453 cells to HGF/SF (100 ng/ml x 20 min) causes a large increase in the degree of activation (tyrosine phosphorylation) of c-Met (Fig. 1A)
. These findings support the role of the c-Met receptor in HGF/SF-mediated cell protection.
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. Gene products whose expression was consistently increased in [HGF/SF+ADR]-treated cells, relative to cells treated with ADR alone, by an average ratio of >1.7 in at least two of three completely independent experiments (i.e., separate cell treatments, RNA isolations, and microarray hybridizations) are listed in Table 3
. Those gene products for which expression was consistently decreased in cells treated with [HGF/SF+ADR] relative to ADR alone (ratio <0.7 in at least two of three completely independent experiments) are listed in Table 4
. The ratio values shown in these tables represent the mean ± range (n = 2) or mean ± SD (n = 3). Some of the cDNA sequences contained on the microarray slides corresponded to expressed sequence tags for which the full-length sequence is not available in public domain databases. Alterations in the expression of cDNAs corresponding to these cDNAs, for which there is little or no information available on the structure-function of the putative gene product, are not included in Tables 3
and 4
.
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). The abbreviations for these gene products are defined, and their functions (or putative functions) are shown in Table 3
. Genes for which the mRNA levels were reproducibly decreased in [HGF/SF+ADR]-treated cells (relative to ADR alone) included those in similar functional classes: including DNA damage response (e.g., TOPBP1), cell cycle regulation (e.g., c-Myc and CIP-4), signal transduction (e.g., 51C and STK2), and protein and RNA metabolism (e.g., human Gu protein). Few or no gene products for which expression was reduced were observed in several functional classes, including development and differentiation, transcriptional regulation, and general cellular metabolism. However, in interpreting the significance of the lack of genes whose expression was decreased in certain functional classes, it should be noted that: (a) the number of genes included in each functional class is influenced by the ratio cutoffs, which is arbitrary; (b) fewer genes showed decreased than increased expression, based on the ratio criteria chosen; and (c) the inclusion of genes in the different functional categories was somewhat arbitrary, because some genes could be included in more than one category.
Table 5
shows a cDNA microarray comparison of gene expression in MDA-MB-453 cells treated with HGF/SF relative to untreated control cells. These data indicate that the number of genes whose expression is reproducibly altered and the magnitude of the alterations are relatively small when the experiment is performed in the absence of treatment with ADR. However, it was noted that 51C (INPPL1), which was decreased in [HGF/SF+ADR]-treated cells relative to ADR alone, was also decreased in HGF/SF-treated cells relative to control.
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and 4
, via semiquantitative RT-PCR assays, using techniques described before by us (12
, 13) . The PCR primers and reaction conditions are provided in Tables 1
and 2
, respectively. For each PCR assay, the reaction conditions and cycle numbers were individually optimized and adjusted so that the reaction fell within the linear range of product amplification. ß-Actin and ß2-microglobulin, two genes whose expression was not altered, were used as controls for loading. The levels of amplified PCR products were quantitated by densitometry and expressed relative to ß-actin. Figs. 2
and 3
show semiquantitative RT-PCR results for genes whose expression was either increased (Fig. 2)
or decreased (Fig. 3)
in cells treated with [HGF/SF+ADR] relative to ADR alone.
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and 3
show 16 different genes for which expression was either increased (n = 7) or decreased (n = 9) in [HGF/SF+ADR]-treated cells by both cDNA microarray and semiquantitative RT-PCR analyses. Genes confirmed to be increased in the [HGF/SF+ADR] group included: ATM (ataxia-telangiectasia mutated), PKD1 (polycystic kidney disease-1), lysyl hydroxylase, LDH-A (lactate dehydrogenase-A), U1 snRNP70 (U1 small nuclear riboprotein, Mr 70,000), VEGF (vascular endothelial growth factor), and PGK1 (phosphoglycerate kinase). Genes confirmed to be decreased in the [HGF/SF+ADR] group included: c-Myc, CIP4 (cdc42-interacting protein-4), S100A9 (calgranulin), B94 (a TNF-inducible gene product), 51C (an inositol polyphosphate-5-phosphatase, also known as INPPL1 and SHIP-2), TOPBP1 (a DNA topoisomerase IIB binding protein), STK2 (a serine/threonine protein kinase), PTPN2 (a protein tyrosine phosphatase), and Gu protein (an RNA helicase).
Some of these alterations, although novel and not otherwise predictable, make sense within the context of explaining how HGF/SF may protect DNA-damaged cells, as will be considered in depth in the "Discussion." The down-regulation of 51C in [HGF/SF+ADR]-treated cells was of particular interest because: (a) a decrease in 51C mRNA levels was also noted in cells treated with HGF/SF alone (related to sham-treated control cells); and (b) 51C is a lipid phosphatase, analogous to PTEN, except that 51C removes the 5-phosphate whereas PTEN removes the 3-phosphate (14)
. Thus, 51C, similar to PTEN (15)
, might be expected to inhibit c-Akt activation (see "Discussion"). Thus, we also examined 51C expression levels by semiquantitative RT-PCR using a completely different set of primers. Similar results were obtained for 51C using both sets of PCR primers (see Fig. 3
).
Finally, it is noted that the RT-PCR assays provide additional information not obtained in the microarray comparisons. The RT-PCR assays allow comparisons of gene expression in cells treated with ADR, relative to control, a comparison not made by cDNA microarray analysis. Thus, in Fig. 2
, it was observed that in most cases, the main effect of HGF/SF was not to alter gene expression by itself but to block the ADR-induced reduction of mRNA levels that were observed in the absence of HGF/SF. In Fig. 3
, with the exception of 51C and PTPN2, HGF/SF by itself did not significantly alter gene expression; but its main effect was to block the ADR-induced up-regulation of mRNA levels. However in some cases, the mRNA levels in [HGF/SF+ADR]-treated cells were reduced to below control levels (e.g., CIP4 and TOPBP1).
Role of CIP4 in HGF/SF-mediated Protection against ADR.
The cdc42-interacting protein-4 (CIP4) was originally identified as a protein that binds to the activated form of cdc42, a Rho-like small GTPase, and was subsequently found to bind to the Wiskott-Aldrich syndrome protein (WASP) through its COOH terminus and to microtubules through its NH2 terminus (Refs. 10
, 16
; illustrated in Fig. 4A
). Although CIP4 is not known to be involved in cell survival or apoptosis pathways, the finding that CIP4 mRNA expression is up-regulated by ADR and that HGF/SF blocks the ADR-induced up-regulation of CIP4 raises this possibility. To determine whether CIP4 could modulate the survival of MDA-MB-453 cells in response to of ADR or HGF/SF, MDA-MB-453 cells were transfected with expression vectors encoding wild-type (wt) or mutant (truncated or deleted) forms of CIP4 containing an NH2-terminal myc epitope tage and then assayed for their survival response. The MTT assay, which measures cytotoxicity as the loss of mitochondrial function (i.e., the ability to reduce a tetrazolium dye to formazan) was used to quantitate cell viability (11)
. Expression of these proteins was confirmed by Western blotting of transfected cells using an anti-myc antibody (see Fig. 4B
).
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, consistent with a role as a modulator of DNA damage or apoptosis response pathways. In the absence of HGF/SF, the decrease in cell survival (viability) in wtCIP4-transfected cells (relative to the empty vector transfected control) treated with ADR alone was greater at 10 µM ADR (-28%; P < 0.001, two-tailed t test) than at 20 µM ADR (-10%; P < 0.05, two-tailed t test). This finding might reflect a greater degree of up-regulation of endogenous CIP4 expression at the higher dose of ADR, so that the transfected wtCIP4 has a smaller effect. For cells treated with HGF/SF, at both 10 and 20 µM ADR, the survival of the wtCIP4-transfected cells was significantly lower than the empty vector-transfected cells (P < 0.001).
The quantitative degrees of cell protection by HGF/SF were calculated based on the following equation, where (S/So) = cell viability relative to control:
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. On the basis of these calculations, transfection of wtCIP4 reduced the HGF/SF- mediated cell protection from 
85 to 40%. On the other hand, there was no effect of wtCIP4 on cell viability in the absence of ADR (100% of control).
Expression vectors encoding mutant forms of CIP4 included a deletion of the microtubule binding domain (CIP4 118–545), a deletion missing the cdc42 binding region (CIP4 
383–481) and a deletion of the COOH-terminal WASP binding domain (Fig. 4A)
. In general, these deletion mutants had little or no effect on the degree of HGF/SF-mediated cell protection, nor did they affect cell viability in the absence of ADR (Fig. 4C)
. However, cells transfected with the mutant CIP4 cDNAs did show an increase in cell viability (by 
15–20%) at 20 µM ADR in the absence of HGF/SF. This finding may be attributable to their function as dominant inhibitors of the endogenous wild-type CIP4, although that conclusion cannot be made from this experiment alone.
Similar findings were obtained using another cell type that is also protected against ADR-induced DNA damage by preincubation with HGF/SF, DU-145 human prostate cancer cells (6) . Thus, wtCIP4, but not the mutant or truncated forms of CIP4, blocked the HGF/SF-mediated protection against ADR (data not shown). These findings are consistent with a role for CIP4 as a regulator or modulator of cell survival in the setting of DNA damage.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have reported previously that in addition to protecting cells against cytotoxicity and apoptosis induced by DNA damage, HGF/SF enhanced the ability of carcinoma cells, including MDA-MB-453 cells, to repair DNA strand breaks induced by ADR or X-rays (6) . The observation that cells treated with [HGF/SF+ADR] show altered expression of certain gene products involved in DNA damage response pathways is consistent with that prior finding. For example, ATM (ataxia-telangectasia mutated), a nuclear protein kinase involved in DNA damage signaling (17) , and FEN1 (flap endonuclease-1), an enzyme implicated in the base excision repair pathway (18) , were up-regulated in [HGF/SF+ADR]-treated cells. A mutation or deletion of the ATM gene leads to a defect in the repair of double-strand DNA breaks and increased sensitivity to ionizing radiation.
We also found that ADR caused the down-regulation of the PKD1 (polycystic kidney disease-1) gene product, and HGF/SF blocked the ADR-induced down-regulation of PKD1 expression. PKD1 has been identified as a developmentally regulated gene, the absence of which is linked to type I autosomal dominant polycystic kidney disease (19) . The function of this gene is not well understood, but PKD1 was found to encode a large cell membrane protein associated with the cadherin-catenin cell:cell adhesion complex (20) . Interestingly, the PKD1 gene product was shown recently to play roles in maintaining the structural integrity of blood vessels (21) and in protecting MDCK epithelial cells against apoptosis (22) . We had reported previously that HGF/SF protects both vascular endothelial and MDCK epithelial cells against DNA damage-induced apoptosis (4 , 5) . Thus, inhibition of the down-regulation of PKD1 by HGF/SF may be a cytoprotective function, one which merits further investigation.
On the other hand, the expression of the topoisomerase binding protein TOPBP1, which binds DNA topoisomerase IIB and also shows DNA strand break binding activity (23, 24, 25) , was decreased in cells treated with [HGF/SF+ADR]. ADR causes DNA strand breakage in part by converting the DNA topology enzyme topoisomerase II into a DNA cleaving enzyme (26) . It is thought that topoisomerase binding proteins such as TOPBP1 may contribute to or potentiate ADR-mediated DNA damage, but the role of TOPBP1 in this process remains to be established. The finding that ADR up-regulates TOPBP1 expression and that the up-regulation is blocked by HGF/SF is provocative, because it suggests a potential mechanism by which HGF/SF might modulate the DNA damage and repair process, upstream of DNA-damage induced apoptosis. HGF/SF blocked the ADR-induced up-regulation of the human Gu protein. Gu is a DEXD box nucleolar RNA helicase, which presumably participates in aspects of RNA synthesis and processing (27) . This finding is interesting because recent evidence suggests that, like topoisomerase II, Gu may be a target of ADR (28) . However, the significance of this finding relative to HGF/SF-mediated cell protection remains to be determined.
A number of gene products implicated in signal transduction pathways were found to be up-regulated (e.g., RhoB and RAB5A) or down-regulated [e.g., STK2 (a serine/threonine kinase), PTPN2 (also known as T-cell protein tyrosine phosphatase, TCPTP) and 51C (also known as INPPL1 or SHIP-2)]. Expression of the 51C gene, which encodes an the inositol polyphosphate-5-phosphatase (29)
, was decreased in both HGF/SF-treated cells (relative to control) and [HGF/SF+ADR]-treated cells (relative to ADR alone). This finding is of particular interest because of previous studies demonstrating a requirement for P13K c-Akt signaling in the HGF/SF-mediated protection of breast cancer (MDA-MB-453) and glioma cell lines against apoptosis (6
, 7
, 30)
.
It had been reported previously that the tumor suppressor PTEN/MMAC1, an inositol polyphosphate-3-phosphatase, inhibited the PI3K/Akt pathway through its lipid phosphatase activity (15) . Recently, 51C was similarly found to act as an inhibitor of the PI3K/Akt pathway, presumably also by reducing the levels of phosphatidylinsitol-3,4,5-phosphate [PI(3,4,5)P3], which is generated through the lipid kinase activity of PI3K (31) . Thus, the reduced expression of 51C in HGF/SF-treated cells should have the effect of maintaining the levels of PI(3,4,5)P3, which is essential for the activation and proper localization of c-Akt.
Interestingly, it has been demonstrated that one of the splice variants of the protein tyrosine phosphatase PTPN2/TCPTP, TC45, can inhibit epidermal growth factor receptor-mediated activation of PI3K/c-Akt signaling (32) . Although the role of PTPN2 in c-Met receptor signaling and the important in vivo substrates for PTPN2 are unclear, the finding that HGF/SF down-regulates PTPN2 gene expression again raises the possibility that PTPN2 is a target for the HGF/SF-mediated protection against DNA-damaging agents.
A cytoskeleton-associated cdc42-interacting protein, CIP4, was found to be up-regulated in ADR-treated cells, whereas HGF/SF blocked the up-regulation of CIP4. The function of CIP4 has not been established definitively, but CIP4 may function, in part, to carry the Wiskott-Aldrich syndrome protein (WASP), a multidomain protein involved in cytoskeletal organization, from actin filaments to microtubules (10) . We showed that forced expression of wild-type human CIP4 reduced the degree of HGF/SF-mediated protection of MDA-MB-453 cells to 50% or less of that observed in untransfected or empty vector-transfected control cells. On the other hand, expression of internally deleted or truncated CIP4 proteins did not inhibit cell protection. These findings suggests a role for CIP4 in cell survival/apoptosis pathways, a finding that is not obvious based on its known activities and protein interactions.
Although we have focused on some of the more novel findings of this study, not all of the cDNA microarray and RT-PCR results were unexpected. For example, the finding that ADR up-regulates c-Myc mRNA expression and that the up-regulation was blocked by HGF/SF was not unexpected. We reported similar results based on Western blotting of MDA-MB-453 cells (5) . The transcription factor c-Myc has been implicated in a variety of cellular processes, including proliferation, differentiation, transformation, and apoptosis. Overexpression of c-Myc renders cells more susceptible to apoptosis through both p53-dependent and p53-independent mechanisms (33 , 34) . Thus, theoretically, down-regulation of c-Myc by HGF/SF in the setting of DNA damage might be expected to confer protection against apoptosis.
We had also reported that ADR down regulates the protein levels of the antiapoptotic protein Bcl-XL, whereas HGF/SF blocks the ADR-induced downregulation of Bcl-XL protein in MDA-MB-453 cells (5)
. Bcl-XL was not present among the cDNAs spotted onto the microarrays slides used in this study. However, we examined the Bcl-XL mRNA expression by semiquantitative RT-PCR analysis and found no ADR or HGF/SF alterations in Bcl-XL mRNA levels in multiple repeat assays.(6)
Thus, the alterations in Bcl-XL protein levels probably occur through translational or posttranslational mechanisms. This finding suggests that some of the protection conferred by HGF/SF may be attributable to alterations in protein processing and metabolism. We had also noted that cell protection required a relatively long preincubation with HGF/SF of 
24 h for some protection and 48 h for maximal protection (5)
. This consideration suggests that the ability of HGF/SF to block the reduction of Bcl-XL protein levels induced by ADR might be attributable to alterations in the expression of genes involved in the processing or metabolism of Bcl-XL.
Our findings suggest the viability of the cDNA microarray approach, coupled with additional studies to confirm gene expression alterations and functional studies to evaluate the significance of the findings, as a means of identifying novel and interesting genes that may be involved in HGF/SF cell protection pathways. It is likely that some of the genes for which expression was altered by HGF/SF in the setting of DNA damage are not involved in cell survival or apoptosis pathways. Alterations in these gene products may reflect other activities of HGF/SF than promotion of cell survival or may be a passive consequence of cell survival rather than a cause of survival. On the other hand, it is also likely that genes not implicated previously in cell survival or apoptosis mechanisms will be found to play roles in these processes (e.g., CIP4).
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FOOTNOTES |
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1 Supported, in part, by USPHS Grants R01-ES09169, R01-82599, and RO1-80000; United States Army Breast Cancer grant DAMD17-99-1-9254; and the Susan G. Komen Breast Cancer Foundation. 
2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, Long Island Jewish Medical Center, The Long Island Campus for the Albert Einstein College of Medicine, 270-05 76th Avenue, New Hyde Park, NY 11040. Phone: (718) 470-7386; Fax: (718) 470-9756; E-mail: erosen{at}lij.edu
3 The abbreviations used are: HGF/SF, hepatocyte growth factor/scatter factor; ADR, Adriamycin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; IP, immunoprecipitation; PI3K, phosphatidylinositol 3-kinase; RT-PCR, reverse transcription-PCR.
4 Internet address: eisen@genome.stanford.edu, 1998–1999.
Received 5/17/01. Accepted 8/30/01.
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 J. C. Mallory, G. Crudden, A. Oliva, C. Saunders, A. Stromberg, and R. J. Craven A Novel Group of Genes Regulates Susceptibility to Antineoplastic Drugs in Highly Tumorigenic Breast Cancer Cells Mol. Pharmacol., December 1, 2005; 68(6): 1747 - 1756. [Abstract] [Full Text] [PDF]
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 M. Jin, J. Yaung, R. Kannan, S. He, S. J. Ryan, and D. R. Hinton Hepatocyte Growth Factor Protects RPE Cells from Apoptosis Induced by Glutathione Depletion Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 4311 - 4319. [Abstract] [Full Text] [PDF]
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 J. Xu, S. Fan, and E. M. Rosen Regulation of the Estrogen-Inducible Gene Expression Profile by the Breast Cancer Susceptibility Gene BRCA1 Endocrinology, April 1, 2005; 146(4): 2031 - 2047. [Abstract] [Full Text] [PDF]
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 J. Jurvansuu, K. Raj, A. Stasiak, and P. Beard Viral Transport of DNA Damage That Mimics a Stalled Replication Fork J. Virol., January 1, 2005; 79(1): 569 - 580. [Abstract] [Full Text] [PDF]
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 I. Bae, S. Fan, Q. Meng, J. K. Rih, H. J. Kim, H. J. Kang, J. Xu, I. D. Goldberg, A. K. Jaiswal, and E. M. Rosen BRCA1 Induces Antioxidant Gene Expression and Resistance to Oxidative Stress Cancer Res., November 1, 2004; 64(21): 7893 - 7909. [Abstract] [Full Text] [PDF]
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