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Anti-Cancer Drug Discovery/Development Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As a starting point for such identification, examination of the role of mitogen-activated protein kinases (MAPKs; i.e., erks, jnks, and p38) and of akt is appropriate because they are key downstream mediators in the signaling cascades activated via trophic ligand receptor interactions for survival and proliferation in other cell types (7 , 8) . In addition, these kinases are expressed in both normal and malignant PrECs (9 , 10) . The activation of akt is significantly increased in primary human tumors, particularly those representing late-stage cancers; this is associated with the loss of PTEN and p27 kip protein expression (10, 11, 12, 13, 14) . The activation of erk and p38 is detectable in premalignant lesions and/or early-stage cancers and, hence, may play a role in the genesis or early progression of prostatic disease (10 , 11 , 15, 16, 17) . Although elevated levels of phosphorylated (i.e., activated) erk have been reported in advanced disease (16 , 18) , additional studies indicate that erk activity declines with disease progression (10 , 11 , 17) and that constitutive erk activity inhibits prostate cancer proliferation (19) . Activated jnk is detectable in the nuclei of basal epithelial (i.e., TA) cells in the normal prostate, and there is a quantitative increase in activated (i.e., nuclear) jnk in prostatic cancer cells (9 , 18) .
Although these correlative studies are important, they do not resolve the causal versus associated nature of the activities of akt and MAPK in androgen-independent prostate cancer cells. To evaluate the causal role of these signaling pathways, appropriate in vitro test systems are required. As an appropriate test system, we characterized the role of akt and MAPK pathways in the in vitro survival of normal prostatic transit-amplifying cells. This is because TA cells, although being ligand (i.e., androgen) independent, do require andromedin signaling for their in vitro survival and proliferation (20) and also because their transformed progeny give rise to prostate cancer (2) . These results were compared with those from a series of five androgen-independent prostate cancer cell lines to identify whether similar or alternative signaling pathways are consistently involved with malignant versus normal prostatic cell survival and proliferation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Stock solutions of 20 mmol/L U0126 (Promega Corp., Madison, WI), 10 mmol/L SB203580 (LC Labs, Woburn, MA), 100 mmol/L SP600125 (Calbiochem, San Diego, CA), and 20 mmol/L Akt inhibitor (Calbiochem) were prepared in DMSO and were stored as per manufacturer’s instructions. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Casodex was kindly provided by AstraZeneca Pharmaceuticals (Cheshire, UK).
Cell Growth Assays.
Live cells were quantitated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (CellTiter 96 Non-Radioactive Cell Proliferation Assay from Promega Corp.), as described previously (20)
. Cell numbers were determined by extrapolation from standard curves of absorbance versus cell number. The number of cells estimated after each treatment was normalized to the starting cell number (1,500 malignant cells per well or 3,200 normal cells per well) and represented as 100%. Data shown are means ± SEM of three independent experiments.
Western Blotting.
Western blots were performed with 50 µg of total protein except in the detection of p38, for which 100 µg were used. Primary antibodies to phospho-erk1/2 (E10 monoclonal), phospho-p38MAPK (polyclonal), phospho-Aktser473 (polyclonal), total erk1/2 (polyclonal), total p38MAPK (polyclonal), and total akt (polyclonal) were purchased from Cell Signaling Technology (Beverly, MA); p27 kip and p21cip monoclonal antibodies were purchased from BD Biosciences (San Diego, CA); Rb (C-15), p53 (FL-393), phospho-c-jun (serine 73-R), total c-jun (H-79), and androgen receptor (N-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); PTEN (clone 6H2.1) from Cascade Bioscience (Winchester, MA); and involucrin (clone 545) and transglutaminase II (clone TG-100) from Neomarkers, Inc. (Freemont, CA). Blots were routinely stripped in buffer containing 1% ß-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl (pH 6.7) for 1 hour at 37°C and reprobed as mentioned in the figure legends.. Secondary horseradish peroxidase-conjugated antibodies and chemiluminescent detection reagents (ECL) were purchased from Amersham Biosciences Corp., Piscataway, NJ. Quantitative estimation of band intensity was determined by densitometry with Kodak Digital Science software. The ratio of the amount of phosphorylated protein to the amount of total protein was calculated in each case.
Determination of Percentage of Proliferating Cells.
Cytospun (100 µl of 2 x 105 cells/ml suspension) cultures were fixed in 3% paraformaldehyde (4°C for 30 min) and were stained with an antibody directed against Ki67 (Immunotech Inc., Marseille, France) at a dilution of 1:50, as described previously (3)
. The percentage of cells in S phase was assayed by bromodeoxyuridine (BrdUrd) incorporation in the 5-bromo-2'-deoxyuridine Labeling and Detection Kit II from Roche (Indianapolis, IN), as described by the manufacturer. Mean results were obtained from three experiments, each being scored from a minimum of three fields.
Determination of Percentage of Apoptotic Cells.
Seventy percent-methanol-fixed cells were rehydrated and stained with a 1.5 µg/ml concentration of 4',6-diamidino-2-phenylindole (DAPI) in phosphate buffer saline (Vector Laboratories, Burlingame, CA). The stained cells were observed under a Zeiss UV-fluorescent microscope for apoptotic morphology, as described previously (21)
. Mean results were obtained from three experiments, each being scored from a minimum of three fields.
Time-Lapse Videomicroscopy.
T-25 flasks seeded with 6 x 105 cells were imaged using phase contrast optics and were evaluated for proliferation and apoptosis, as described previously (21)
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In addition, all of these cancer cell lines are androgen independent (i.e., do not require androgen in the medium for their survival and proliferation) and, thus, are the type of cells that are ultimately lethal to prostate cancer patients (1)
. Although androgen independent, two of these lines (LAPC-4 and CWR22Rv1) still retained a limited sensitivity to androgens as documented by the ability of the anti-androgen Casodex, at concentration 10 µmol/L, to inhibit proliferation when added to the optimal growth medium, even though it did not induce apoptotic death of either cell line (Table 3)
. In contrast, the remaining lines [i.e., in late passage (>100 passages) LNCaP (termed LNCaP-LP), PC-3, and DU-145] were androgen independent and had no response to Casodex (Table 3)
. In addition, because normal and malignant prostate epithelial cells both produced high-growth fraction (i.e., >60% Ki67-positive) cultures in vitro (Table 1)
, valid comparisons can be made about the importance of specific downstream kinase signaling in their growth regulation.
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. These results documented that proliferating normal prostate TA cells had detectable steady-state levels of active erk1 and 2, p38, c-jun, and akt. These results were compared with those obtained using the five prostatic cancer cell lines. To do this, we normalized the results to the level of kinase activity observed in normal prostatic transit-amplifying (PrEC) cells (Fig. 1)
under optimal growth conditions (i.e., in the presence of growth factors) as determined by Ki67 expression (Table 1)
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, even though they all had comparable growth and S-phase fractions (Table 1)
. In PC-3 cells, p38 activity was comparable with that in normal PrECs, although p38 activity was reduced in the other four malignant cell lines (Fig. 1B)
. Jnk activity, as assayed by phospho-c-jun levels, was significantly increased in all of the malignant lines, compared with normal cells (Fig. 1C)
. Akt activation was higher in the PTEN mutant LNCaP and PC-3 cells, as compared with normal PrECs (Fig. 1D)
. Akt activity was lower, however, in DU-145 and CWR22Rv1 cells (i.e., both PTEN wild-type) and equal in LAPC-4 cells than in normal PrECs (Fig. 1D)
. The above results documented that there were consistent differences in the steady-state levels of activation of the various MAPKs in malignant (lower erk and p38 activity, and higher jnk activity) versus normal prostate cancer cells, even though the cells were matched for growth and S-phase fractions.
Differential Response of the Akt and MAPKs to Growth Fraction Deprivation in Normal versus Malignant Prostate Cells.
The high rate of proliferation of normal TA cells was rapidly decreased (P < 0.05) after removal of the exogenous growth factors (Table 1)
. After a 3-day period of growth factor withdrawal, the number of viable normal cells remained constant (20)
, although the growth fraction (i.e., the percentage of Ki67 positive) and S-phase fractions (i.e., the percentage of BrdUrd incorporation positive) decreased (P < 0.05) by more than 70% (Table 1)
. At this 3-day deprivation time point, akt activation was decreased by 2- to 3-fold, phosphorylated p38 and jun levels (i.e., jnk activity) remained unchanged, and phosphorylated erk 1/2 levels were twice as high, as assayed by the densitometric levels of phosphorylated protein normalized to that of ß-actin (Fig. 2)
. Expression of p21cip and p27kip in these growth factor-deprived normal cells was tested, because these molecules can inhibit entrance into the cell cycle by their binding to cdk2/cyclin E and other cdk/cyclin complexes (23)
. Whereas such growth factor deprivation decreased the proliferation of PrECs (Table 1)
, it did not induce any change in p21cip protein nor induce p27kip protein expression (Fig. 2)
. These results further suggested that survival of normal TA cells was provided by cell-attachment-dependent signaling, although proliferation requires additional growth factor-dependent signaling.
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. In contrast to normal prostatic cells, however, p27kip was expressed by all of the malignant prostatic cell lines, although only two of five lines (i.e., CWR22Rv1 and LNCaP-LP) expressed p21cip during high-growth-fraction growth in the presence of growth factors (Fig. 2)
. To deplete the levels of autocrine growth factors, malignant cell lines were washed and fresh medium that did not contain androgen or serum was replaced daily. After 3 days of such growth factor deprivation, the growth and S-phase fraction of each of the malignant lines was significantly (P < 0.05) reduced (Table 1)
with no indication of an enhancement in apoptosis as assayed by increased DAPI staining or decreased viability using MTT assays (data not shown). Thus, like the situation for normal prostatic cells, malignant cell attachment are sufficient to induce survival signaling, although continuing to require growth factor input for proliferation. Unlike the situation for normal prostate cells, however, associated with this decrease in growth fraction was an up-regulation in p27kip protein in growth factor-deprived cultures for all of these malignant lines, although p21cip expression was enhanced only in LNCaP-LP and PC-3 cells (Fig. 2)
. In CWR22Rv1 cells, p21cip was expressed but it did not increase after growth factor removal (Fig. 2)
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Also, unlike the situation for normal prostate cells, 3-day growth factor deprivation did not result in an increase but, instead, in a decrease in erk 1 and 2 activity in all of the malignant lines except PC-3. Growth factor deprivation increased p38 activation in LNCaP-LP, LAPC-4, and PC-3 cells but decreased it in DU-145 cells, with little expression in CWR22Rv1 cells (Fig. 2)
. As in normal cells, akt activation is lowered by 2- to 4- fold in PTEN wild-type, CWR22Rv1, DU145, and LAPC-4 cells. Changes in akt phosphorylation were not detectable in PTEN loss of function LNCaP-LP and PC-3 cells (Fig. 2)
. Unlike normal cells, jnk activation, as assayed by phospho-c-jun, was decreased by growth factor deprivation in all of the malignant cells (Fig. 2)
. These results demonstrated that although growth factor deprivation did not induce apoptosis, it did reduce overall cell proliferation equally in normal and malignant cells (Table 1)
. This deprivation had differential effects on erk, p38, and jnk activity (Fig. 2
; i.e., increased erk, decreased akt activity in normal cells versus increased p38, and decreased erk, jnk, and akt activity in malignant cells).
Kinetics of Akt and MAPK Activation during Cell Cycle Progression in Normal versus Malignant Prostate Cells.
These previous results raised the issue of whether differences in regulation of these activities occurs when cells deprived of growth factors for 3 days are induced to reenter the cell cycle after growth factor replacement. For these studies, CWR22Rv1, LNCaP-LP, and DU-145 lines were used, and the results were compared with those from normal PrECs during growth factor deprivation/restimulation-induced cell cycle recruitment. Under conditions of growth factor deprivation, erk, p38, and jnk activity remained high in normal PrECs, and these levels were not changed by growth factor restimulation (Fig. 3A and B)
. In contrast, erk and p38 activities were low during serum deprivation and were transiently enhanced on restimulation of malignant cells with serum (Fig. 3A and B)
. Densitometric quantitation of Western blots indicated that erk was maximally activated, compared with the growth factor-deprived cultures, by 29-fold (P < 0.05) in CWR22Rv1 at 30 minutes of restimulation, by 7-fold (P < 0.05) in LNCaP-LP at 30 minutes of restimulation, and by 8-fold (P < 0.05) in DU-145 cells at 10 minutes of re-exposure to serum. The maximal erk activation observed in restimulated CWR22Rv1 cells was transitory and monophasic, because no additional peaks of activation were detected at various time points (2, 3, 4, 5, 6, 7, 8, 22, 23, and 24 hours) during restimulation (data not shown). This was consistent with an enhanced erk activity in early G1 phase of cell cycle. An additional peak of erk activation was observed in DU-145 cells after 24 hours of re-exposure to growth factors (Fig. 3A)
. Because the doubling time of these cells in the presence of growth factors was 23 hours, this peak was consistent with activation in the G1 phase of the next replication cycle. Transient G1 phase activation of p38 was also observed in all of the malignant cell lines tested [Fig. 3B
; i.e., by 37-fold (P < 0.05) in CWR22Rv1, by 28-fold (P < 0.05) in LNCaP, and by 3-fold (P < 0.05) in DU-145 cells] within 10 minutes of restimulation (i.e., during early G1 phase of the cell cycle). Growth factor replacement also resulted in a rapid increase in akt activity in the malignant cells; this was not a transient but a sustained 2- to 3-fold elevation over the 24-hour period (Fig. 3C)
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Dose Response Requirements for the Specific Inhibition of Akt and MAPK Signaling.
In these studies, U0126 was used as the inhibitor of MEK1/2 (the upstream activator of the erk), to prevent activation of erk1 and 2 (24)
although SP600125 was used as the inhibitor of jnk (25)
. SB203580 was used as the inhibitor of p38 MAPK (26)
. A thrice-modified phosphatidylinositol analogue was used as the akt inhibitor AIN to prevent translocation of akt to the membrane and, hence, activation by 3-phosphoinositide dependent kinase-1 and -2 (27)
. To develop appropriate regimens for inhibiting the individual MAPKs and akt effectively in prostate epithelial cells, we performed preliminary dose-response studies using CWR22Rv1 cells as a validation system. U0126 successfully inhibited the activation of erk 1/2 in response to growth factor restimulation (Fig. 4A and B)
. At a concentration of 10 µmol/L, U0126 diminished erk activity to a level even lower than that in growth factor-deprived cells (i.e., no stimulation). This compound was specific in that it does not affect p38 activation (Fig. 4A and B)
. SB203580 was highly effective in blocking p38 activation at concentrations of 2 to 20 µmol/L. It diminished erk activation at the higher concentrations of 10 to 20 µmol/L; however, as previously reported (28)
, limiting its use to 2 µmol/L for the specific inhibition of only p38 (Fig. 4A and C)
. At a concentration of 20 µmol/L, SP600125 inhibited Jnk (i.e., jnk 1, 2, and 3) by >90% with less than a 20% inhibition of erk or p38, as previously reported (25)
. The phosphorylation of akt was effectively inhibited by 90% at 16 hours after exposure to a 20 µmol/L concentration of the akt inhibitor-AIN (Fig. 4D and E)
. On the basis of these results, U0126, SB203580, SP600125, and AIN were used at 10, 2, 20, and 20 µmol/L concentrations, respectively.
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. In the presence of erk inhibition via U0126 alone, or Jnk inhibition via SP600125 alone, the numbers of live cells were significantly (P < 0.05) reduced to levels below those normalized to the starting cell number, indicating that cell death occurred (Fig. 5A)
. This cytotoxic response was confirmed by time-lapse videomicroscopy. These studies documented that either erk or jnk inhibition alone caused cessation of cell motility and proliferation. Morphologically, these two treatments induced different changes in cell shape. The inhibition of erk resulted in flattening of the cell (Fig. 5B versus C)
, whereas jnk inhibition resulted in cell shrinkage (Fig. 5B versus 5D)
. In both cases, however, these changes were associated with the death of the cells via cornification [i.e., formation of SDS-insoluble cornified envelopes (ref. 29
; Fig. 5E
). This cornification phenotype was due to cross-linking of the cellular protein involucrin via up-regulation of transglutaminase during apoptotic cell death (30)
. Untreated normal PrECs expressed high levels of both involucrin and transglutaminase (Fig. 5F)
. The detection of both larger and small-sized bands compared with the involucrin monomer (Fig. 5F)
was consistent with a continuous cycle of transglutaminase-induced involucrin cross-linking and degradation, even in untreated normal prostatic epithelium. When these untreated PrECs were exposed to 1% SDS, the cells were completely solubilized, even though they contained detectable levels of cross-linked involucrin. In contrast, when PrECs were induced to die by either erk or jnk inhibition, such enhanced cross-linking resulted in cornified envelopes that were insoluble in 1% SDS treatment (Fig. 5E)
. Interestingly when PrECs were induced to undergo apoptotic death via an increase in the intracellular free calcium [induced by treatment with the SERCA (sarco/endoplasmic reticulum calcium ATPase) pump inhibitor thapsigargin, as described previously (31)
], the cells also shrank, apoptosed, and underwent cross-linking, producing cornified envelopes resistant to 1% SDS solubilization. These combined results demonstrated that formation of cornified envelopes is the characteristic end point for the apoptotic death of these normal transit-amplifying PrECs regardless of the inducing agent. Such death and cornification were not produced when PrECs were exposed to the p38 or akt inhibitors. Akt inhibition alone did result, however, in a complete suppression of net growth of PrECs (Fig. 5A)
. Using both time-lapse videomicroscopy and DAPI nuclear DNA staining, we confirmed that akt inhibition did not induce apoptotic death and cornification of these cells but, instead, prevented their proliferation (Fig. 5A)
. The inhibition of p38 alone had only a modest cytostatic effect (Fig. 5A)
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Variation in the Requirement for MAPKs and Akt Activities in the Proliferation and Survival of Malignant Prostate Epithelial Cells.
Exponentially growing malignant prostate cells were treated with erk, jnk, p38, and akt inhibitors alone or in combination (Figs. 6A–E)
. Whereas control and p38-inhibited CWR22Rv1 cells grew exponentially with an essentially equal rate, those treated alone with either the erk, jnk, or akt inhibitor grew at a reduced rate (P < 0.05; Fig. 6A
). The growth reduction induced by combined treatment with all four inhibitors was significantly greater (P < 0.05) than that produced by any single inhibitor alone (Fig. 6A)
. Morphological and biochemical analyses demonstrated that none of the single or combinational treatments induced cornification as assayed by 1% SDS insolubility. As a single agent, only jnk inhibition increased the extent of apoptosis as assayed by DAP1 nuclear DNA staining (Fig. 6F)
. Thus, the growth retardation induced by erk and akt inhibition in CWR22Rv1 cells was due to a decrease in proliferation, not by induction of cell death (i.e., cytostatic effect). In contrast, jnk inhibition alone not only induced a cytostatic effect but enhanced the percentage of cells undergoing apoptosis. This resulting cytotoxic enhancement was synergized by the inhibition of akt and the other MAPKs (Fig. 6F)
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). For the erk and p38 inhibitors, this growth inhibition was not associated with an increase in apoptosis as assayed by DAP1 staining (Fig. 6F)
. In contrast, jnk or akt inhibition alone resulted in an increase (P < 0.05) in apoptosis (Fig. 6F)
, demonstrating that both jnk and akt activity are critical for survival of LNCaP cells. In contrast, LAPC-4 growth was not affected by p38 inhibition but was growth inhibited (i.e., 2–3-fold reduction) by erk, jnk, or akt inhibitors alone (P < 0.05), compared with controls (Fig. 6C)
. This latter effect was due to decreased proliferation and not to enhanced apoptosis (Fig. 6F)
. The best growth inhibition for LAPC-4 cells was produced by combining all four inhibitors (Fig. 6C)
. Only this combinational approach was able to induce an enhanced level of apoptosis (Fig. 6F)
. The growth of DU145 cells was not significantly inhibited by either erk or p38 blockade (Fig. 6D and F)
. Whereas akt inhibition did inhibit the proliferation of DU145 cells, it did not enhance apoptosis of these cells (Fig. 6F)
. Inhibition of jnk alone did decrease the growth of these cells (Fig. 6D)
, via its ability to increase apoptosis (Fig. 6F)
. This apoptotic response was further enhanced by combination with the other inhibitors (Fig. 6D and F)
. The growth of PC-3 cells was decreased by each of the inhibitors (Fig. 6E)
. This was due to a cytostatic effect with apoptosis of PC-3 cells being enhanced only by the combination of all four inhibitors (Fig. 6F)
. In conclusion, monoinhibition of erk, jnk, or akt produced a reduction in cell number in a majority of the malignant cell lines. However, only jnk inhibition produced an increase in apoptosis. Combinatorial inhibition of erk, p38, jnk, and akt resulted in an enhanced induction of apoptosis and a reduction of growth as compared with the effect of inhibition of any single kinase. Thus, these results demonstrate that erk, p38, jnk, and akt have redundant roles in the regulation of survival of prostate cancer cells. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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What is the mechanism for such enhanced downstream redundancy in survival signaling in malignant versus normal prostate cancer cells? There is a growing body of evidence that androgen receptor in either a ligand-dependent or -independent manner can "cross-talk" with other signaling pathways in prostate cancer cells (6)
. Because normal prostatic TA cells do not express androgen receptor, such "cross-talk" is not possible with the lack of redundancy for the erk and jnk survival signaling in these cells. Such an androgen receptor-dependent explanation is also not consistent, however, with the fact that PC-3 and DU145 prostate cancer cells also do not express androgen receptor but still have enhanced redundancy in survival signaling. An alternative possibility is that during cancer progression, molecular changes occur stochastically because of the inherent genetic instability of prostate cancer cells driven by chronic and/or acute inflammation (32)
. In this inflammatory microenvironment, one of the first and most consistent molecular changes is the methylation of the promoter of the detoxifying enzyme glutathione S-transferase 
isoform (i.e., GST) resulting in a decrease in its expression in prostate cancer (32)
. Such a characteristic decrease in GST expression results in a significant growth advantage (33)
because, now, GST cannot bind to and inhibit jnk activity (34)
. Thus, increased jnk activation via a decrease in GST provides a driving force for the genetic instability of prostate cancer. Any genetic change that enhances the redundancy in downstream effectors of survival pathways would provide a selective growth advantage. Such a process of genetic instability coupled with clonal selection has been documented during progression of prostatic cancer in animal model systems (35)
and could explain why such enhanced redundancy in survival pathways is consistently observed during the progression to an androgen-refractory state.
In conclusion, because of the enhanced redundancy in downstream effectors for survival pathways, only the combined inhibition of akt and MAPK signaling induces sufficient apoptosis to produce a therapeutic effective response in all of the androgen-independent prostate cancer cell lines tested. Such redundancy is not unlimited, however (36 , 37) , and provides the critical rationale for developing combinatorial approaches to signal transduction inhibition. Such combinatorial strategies are currently being tested in vivo in nude mice orthotopically bearing the same panel of androgen-independent human prostate cancers characterized in the present studies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: John Isaacs, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 1650 Orleans Street, CRB-1M41, Baltimore, MD 21231. Phone: 410-955-7777; Fax: 410-614-8397; E-mail: isaacjo{at}jhmi.edu
Received 4/10/04. Revised 6/21/04. Accepted 7/ 2/04.
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kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells. Am J Pathol, 159: 431-7, 2001.This article has been cited by other articles:
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H. Gao, X. Ouyang, W. A. Banach-Petrosky, W. L. Gerald, M. M. Shen, and C. Abate-Shen Combinatorial activities of Akt and B-Raf/Erk signaling in a mouse model of androgen-independent prostate cancer PNAS, September 26, 2006; 103(39): 14477 - 14482. [Abstract] [Full Text] [PDF]
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