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Tumor Biology |
Program in Cellular and Molecular Medicine [V. S., S. B. B.], Oncology Center [V. S., M. W. B., R. K. R., D. R. A., B. D. N., S. B. B., D. W. B.], and Department of Medicine [D. R. A., S. B. B., D. W. B.], Johns Hopkins University School of Medicine, Baltimore, Maryland 21231
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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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) to transactivate target genes including HES1 (2)
. HES3
family members and other targets of Notch signaling serve to down-regulate and counterbalance the effects of myogenic and neurogenic bHLH factors including MyoD and the achaete-scute complex. The net result of Notch signaling frequently is to preserve a small population of uncommitted multipotential cells within a differentiating tissue (3
, 4)
. Truncated, constitutively active forms of Notch have been shown to have oncogenic activity in acute lymphoblastic T-cell leukemia (5) , mouse mammary tumors (6) , and transformed kidney epithelial cells (7) . The in vitro transforming activity of Notch in rat kidney cells requires the cooperation of E1A oncogene and nuclear localization. Notch signaling may have opposite actions on cell growth, depending on poorly understood differences in cellular context. For example, in hematopoietic cell maturation, ligand activation of Notch1 inhibits the capability of myeloid precursor cells to differentiate in response to granulocyte colony-stimulating factor (8, 9, 10) . In B-cell development, chicken Notch1 has been shown to induce growth arrest and apoptosis (11) , whereas active Notch1 induces ectopic T-cell proliferation in the bone marrow (12) .
In the developing lung, Notch 1 and HES1 are strongly expressed in non-NE airway epithelial cells, whereas MASH1 is restricted to clustered PNECs (13 , 14) . In HES1 transgenic knockout mice, PNECs are markedly increased, with a parallel up-regulation of MASH1 (14) . Conversely, MASH1 transgenic knockout mice fail to develop PNECs (13 , 14) . Consistent with a well-established role in inhibiting commitment and differentiation in neuronal precursors, Notch signaling appears to play a critical role in restricting NE cell development within the airway epithelium.
Previous studies have revealed a tight concordance between the NE phenotype in lung cancer and hASH1. Disrupting hASH1 function by antisense treatment in SCLC cells results in attenuation of NE markers (13) . In a transgenic model simulating NE lung carcinogenesis, hASH1, in conjunction with SV40 large T antigen, caused airway epithelial cell proliferation, and the appearance of aggressive NE tumors when targeted to normal pulmonary epithelium (15) . We have previously shown that hASH1 may be transcriptionally down-regulated by HES1 in SCLC (16) . However, little is known about Notch action in lung cancer. It is conceivable that NE lung cancers have dysregulation of the normal mechanisms that normally extinguish hASH1 expression in fully differentiated neural and NE cells. Notch influences on SCLC proliferation are of particular interest, because there is evidence that some NE features may contribute to SCLC growth. In the present study, we explored whether activation of Notch signaling could disrupt hASH1 expression and interfere with growth of cultured SCLC. Overexpression of activated forms of Notch1 and Notch2 in SCLC cells caused profound growth suppression stemming from a G1 cell cycle arrest. The CDKIs p21waf1/cip1 and p27kip1 were up-regulated accompanying the arrest. Parallel to the growth arrest phenotype, activation of Notch signaling in the SCLC cells led to complete repression of hASH1 and induction of pMAPK. Interestingly, although HES1 was induced by Notch proteins, overexpression of HES1 alone could only partially replicate the Notch phenotype, implying an important role for HES1-independent actions of Notch in this model system.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Raf-1:ER cells were maintained and induced with 1 µM ß-estradiol as previously described (17)
.
Recombinant Adenovirus Generation and Infection Procedure.
Production of high titer recombinant adenovirus using the AdEasy system has been described previously (18)
. Briefly, human intracellular Notch1 and Notch2, and HES1 were generated by PCR and subcloned into a pAdTrackCMV shuttle plasmid, which allows bicistronic expression of GFP and the inserted gene under cytomegalovirus promoters. The intracellular Notch1 fragment contains amino acids 1759–2556 and was amplified from plasmid TAN-1/pCDNA3; Notch2 contains amino acids 1700–2471 and was amplified from plasmid cytohN2pcAMP/pcDNAI/AMP (both gifts of S. Artavanis-Tsakonas, Massachusetts General Hospital Cancer Center, Charlestown, MA). The HES1 fragment was amplified from human lung cancer cDNA. Then the shuttle plasmids with inserted genes were cotransformed with pAdEasy1 into BJ5183 bacterial competent cells to generate the recombinant adenoviruses. High titer viral stocks were first transfected and then amplified in 911 cells. The control virus expresses the Escherichia coli ß-galactosidase gene (AdßGal). Each viral stock was titered by plaque assay in low-passage 293 cells. A series of preliminary infections were performed in each cell line to determine the optimal dose of viruses, allowing at least 70% GFP reactive cells with acceptable cytotoxicity determined by growth rate compared with the mock-infected cells. The final doses were 2.5–5 plaque-forming units/cell for DMS53 or 10 plaque-forming units/cell in NCI-H209. In each experiment, the level of GFP expression at 48 h postinfection was assessed to confirm the efficiency of infection.
DNA Sequencing.
Vector constructs were verified by two strands sequencing at the Johns Hopkins Core Sequencing facility. HES1 genomic sequencing was performed with PCR amplification of exons 1–5, containing the entire coding sequence (19)
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Growth Assays.
The colorimetric assay using MTT (Sigma, St. Louis, MO) was performed as described previously (20
, 21)
with some modification. Cells were passaged in phenol red-free RPMI 1640 with 10% FBS and antibiotics, then seeded into 24-well plates at 20,000 and 40,000 cells per well for DMS53 and NCI-H209 cells, respectively. For DMS53 cells, 250 µl of fresh medium containing 10% of MTT 5 mg/ml stock was replaced to each well at the day of harvesting. Plates were incubated at 37°C for 3 h; then 750 µl of DMSO (Sigma) was added to each well and shaken at room temperature for 10 min to dissolve intracellular MTT formazan crystals, followed by absorbance measurement at 540 nm. For NCI-H209 cells, the MTT stock was added at 10% of culture volume for each well, and plates were incubated at 37°C for 4 h. After brief centrifugation, all but 
100 µl of medium was removed, 900 µl of DMSO was added per well, and absorbance was measured at 540 nm. Experiments were done in quadruplicate and were repeated at least twice. Statistical analysis was performed by GraphPad Prism software version 3.02 (GraphPad Software Inc., San Diego, CA) with two-way ANOVA.
Cell Cycle Analysis.
Cells were stained by propidium iodide according to Vindelov’s method (22)
. Briefly, DMS53 or NCI-H209 cells were harvest at 3 or 4 days after infection. Nuclei were prepared, stained with propidium iodide, and subjected to flow cytometric analysis with an EPICS 752 flow cytometer (Coulter Electronics, Hialeah, FL). The cell cycle parameters from 10,000 gated nuclei were determined with multicycle software (Phoenix Flow System, San Diego, CA). Experiments were done at least twice.
Immunoblotting.
Infected cells were harvested at the time indicated in each experiment. Cells were lysed in 1x SDS sample buffer (62.5 mM Tris, 2% SDS, 10% and glycerol) with aprotinin, leupeptin, pepstatin, and 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF). Cell suspensions were briefly sonicated and protein concentration for each cell lysate was determined by DC Protein Assay (Bio-Rad, Hercules, CA) according to the manufacturer’s instruction. Fifty to 100 µg of total protein from whole cell lysate were loaded to each lane during gel electrophoresis. Equivalent loading and transfer were verified by filter staining with Fast Green (Fisher Scientific, Pittsburgh, PA). Western blot analysis was performed by using 0.1 M Tris (pH 7.5), 0.9% NaCl, and 0.05% Tween 20 (TBST) with 5% nonfat dry milk as blocking and antibody dilution buffer. Working concentrations of antisera were as follows: Notch1, 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA); Notch2, 1:50 (anti-bhN6, gift from S. Artavanis-Tsakonas, Massachusetts General Hospital Cancer Center, Charlestown, MA); HES1, 1:10000 (gift from T. Sudo, Toray Industries, Inc., Kanagawa, Japan); MASH1, 1:1000 (BD PharMingen, San Diego, CA); p27, 1:500 (Santa Cruz Biotechnology); p53, 1:1000 (Labvision, Fremont, CA); p16, 1:1000 (Labvision); p21, 1:1000 (Labvision); pMAPK, 1:1000 (New England Biolabs, Beverly, MA); PARP, 1:5000 (BD PharMingen); and G3PDH, 1:5000 (Trevigen, Gaithersburg, MD). A Supersignal West Pico (Pierce, Rockford, IL) chemiluminescence kit was used for all antibodies with the exception that the Supersignal West Femto kit (Pierce, Rockford, IL) was used for antisera to MASH1, p16, and p21.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We previously attempted to create stable cell lines expressing constitutively active Notch1 and Notch2, but noted a strong selection bias against clones expressing the Notch constructs (data not shown). Therefore, we elected to express Notch1 and Notch2 using recombinant adenoviruses. We were able to achieve at least 70% of cells expressing GFP at 48 h postinfection in both cell lines with minimal evidence of cytotoxicity. On adenovirus infection, DMS53 cells expressed GFP as early as 6 h and NCI-H209 cells as early as 24 h (Fig. 1C)
postinfection. Expression of the predicted forms of Notch1 and Notch2 was very robust in both cell lines by immunoblotting (Fig. 1A)
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. The entire coding sequence of the endogenous HES1 gene appeared to be intact in both DMS53 and NCI-H209 cells (data not shown). In NCI-H209, the level of HES1 induced by adenoviral HES1 was comparable with the level of endogenous HES1 induced by AdNotch1 or AdNotch2 (Fig. 1B
, compare Lanes 3–6). NCI-H209 cells normally grow in suspension as tight floating cell clusters. The AdNotch1 caused the cell clusters to adhere to the bottom of the plastic culture vessels. The peripheral cells of the adhering clusters had a tendency to spread out from the clusters with elongated cytoplasmic processes (Fig. 1C)
. The magnitude of changes was maximal beginning at 4 days after infection, when 70–80% of spheroids adhered to the plastic. The AdNotch2 infection also caused similar effects but to a lesser extent than the AdNotch1, with only 10–20% of cells showing the adherent morphology. DMS53 cells, which normally grow as a tightly adherent monolayer, exhibited rounding and detachment from the plastic starting from 24 h postinfection by AdNotch1 and AdNotch2 (Fig. 1C)
. None of these morphological changes were observed with the control virus or with AdHES1.
The growth rates of DMS53 cells infected by AdNotch1 or AdNotch2 markedly decreased compared with the rates from the control virus or the AdHES1-infected cells (Fig. 2A)
. In NCI-H209, AdNotch1 completely arrested cell proliferation (Fig. 2B)
. The effect of AdNotch2 on the growth of NCI-H209 was minimal, paralleling the weaker morphological effect of Notch2 in this cell line. In both cell lines, the effect of HES1 was indistinguishable from that of cells expressing the ß-Gal control virus. To screen for a nonspecific viral cytotoxic effect, we compared the growth rate of mock-infected and control virus (AdßGal)-infected cells. We observed a small but statistically significant decrease of growth rate attributable to virus alone (Fig. 2, A and B)
.
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; Table 1
). Although Notch1 exerted a stronger growth arrest effect than Notch2 in DMS53 cells by MTT, we were unable to detect a significant difference in their potency to induce the G1 arrest at 72 h after infection (compare Table 1
and Fig. 3
with Fig. 2
results). In contrast to the reported induction of apoptosis by Notch1 in B cells (11)
, we could not observe a significant sub-G0-G1 peak in our study, which suggested that there was not a significant fraction of apoptotic nuclei at that time point. Moreover, bis-benzimide trihydrochloride (HOECHST 33342; Sigma) nuclear staining and immunoblotting to detect a PARP cleavage product did not reveal an obvious increase in apoptosis (data not shown).
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, p21, Lanes 5 and 6). The levels of p27kip1, readily detectable in these cells, did not increase with Notch proteins. Activated Notch did not induce p16 or p53, nor did it down-regulate cyclinD1 or cyclinE (Fig. 4A
, p53, p27, p16; data not shown for cyclinD1 and cyclinE immunoblotting). In NCI-H209 cells, which have an inactive mutant Rb gene (24)
, AdNotch1 caused a significant increase of both p21waf1/cip1 and p27kip1 corresponding to the G1 arrest, whereas the control viral infection did not cause induction of the CDKIs (Fig. 4B
, compare Lanes 6–9 versus 2–5). Significantly, adenoviral HES1 was unable to induce growth arrest, cell cycle changes, or CDKI expression in either cell line.
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, Lanes 3–6, and 5B
, Lanes 6–9). In contrast, overexpression of HES1 caused a more modest and slower paced decrease in hASH1 protein abundance (Fig. 5A
, Lanes 7–9). These data correlate well with the slow decline observed in hASH1 mRNA over 96 h when we used an inducible rat HES1 vector in DMS53 cells (16)
. The magnitude of reduction of hASH1 expression corresponded well with the extent of the morphological changes and the growth arrest in SCLC cells infected by the Notch adenoviruses.
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. Strikingly, Notch overexpression in both of the SCLC cell lines led to an increase in pMAPK, corresponding to the growth arrest phenotype (Fig. 6B
, Lanes 4–5, 11–13). Thus, in DMS53 cells, Notch1 markedly increased phosphorylated ERK1 and ERK2, whereas Notch2 had a weaker but still significant effect. In NCI-H209 cells, only Notch1 could induce MAPK activation. Treatment with the dual MEK inhibitor, U0126, resulted in toxicity in both DMS53 and NCI-H209 cells. Therefore, we were unable to definitively determine whether MAPK phosphorylation is necessary for the Notch-induced cell cycle block in these cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a limited number of tumor types, including human T-cell leukemia and mouse mammary tumors, Notch signaling is oncogenic rather than antiproliferative (5 , 6 , 26) . However, a recent study showed opposite functions of Notch depending on the cell context. In the development of lymphoid cells, Notch promotes T-cell proliferation but induces apoptosis and growth arrest of B-cell precursors (11) . The cellular mechanisms that mediate growth arrest by Notch remain unclear. It is unlikely that an intact Rb pathway is necessary for Notch-induced arrest because this effect occurred in NCI-H209 cells, which have an inactive mutant Rb gene. The inability of HES1 to induce growth arrest indicates the contribution of HES1-independent mechanism(s). We observed consistently distinct responses to Notch1 versus Notch2 in the SCLC cells. With careful titering and expression studies, we believe that the differences stem from an intrinsically different response to the two forms of Notch rather than a bias in protein dosage. Interestingly, Notch2 was also less potent than Notch1 in inducing transformation of E1A-immortalized rat kidney cells (7) .
Activation of the ras/raf/MAPK pathway is frequently associated with transformation of primary cells and tumor progression of many established cancer cells (27 , 28) . However, an increasing body of evidence supports the idea that constitutive activation of this pathway may lead to growth arrest and terminal differentiation in some cellular contexts. This phenomenon has been observed in SCLC (17 , 25) and medullary thyroid cancer cells (29) , as well as in PC12 rat pheochromocytoma cells, (30) for example. In certain developmental contexts, there is evidence of an antagonistic interaction between Notch and ras signaling in a cell-autonomous manner (31, 32, 33) . However, a recent study in mammary tumors that were derived from transgenic activated Notch4 mice showed a possible cooperation between ras signaling and Notch-mediated tumorigenesis (34) . Although our findings indicate a positive interaction between the two signaling pathways, the cell cycle arrest induced by Notch may not be solely dependent on the MAPK activation. Under our experimental conditions, we have been unable to demonstrate reversal of this growth arrest phenotype by MEK inhibitors. Interestingly, the induction of pMAPK coincided well with the hASH1 reduction and cell cycle arrest by Notch. This association may represent a possible cooperative interaction between the Notch and ras signaling pathways in controlling growth and NE differentiation in SCLC cells. It will be of great interest to elucidate the exact mechanisms of the cross-talk between the two signaling pathways, which may potentially be applicable to other cell contexts.
Our data showed a correlation between the cell cycle arrest by Notch signaling and the induction of p21waf1/cip1 in two different SCLC lines with either wild-type or mutated Rb. Both of these cell lines have an intact p53. The potential interaction between Notch signaling and p21waf1/cip1 is fascinating in light of developmental parallels. Notch signaling is a hallmark of quiescent, multipotent cells in hematopoietic (35 , 36) and central nervous system development (37 , 38) , for example. In the maturation process of these tissues, activation of Notch signaling blocks the capability to respond to differentiating stimuli (39, 40, 41) . A recent study showed that p21waf1/cip1 plays a crucial role in maintaining the self-renewing properties of hematopoietic stem cells (42) . It will be interesting to determine whether Notch functions in maintaining multipotent progenitor cells through p21waf1/cip1.
In summary, Notch signaling in cultured SCLC cells results in a profound G1 cell cycle arrest associated with p21waf/cip1 induction, repression of hASH1, and induction of the downstream ras signaling pathway. Significantly, the current study suggests a direct link between Notch and the raf/MEK/MAPK pathway, although it is presently unclear at what level the MAPK pathway is being activated and what effectors downstream of Notch are implicated. In future studies, it will be important to characterize the signaling pathway responsible for Notch-dependent growth arrest in SCLC cells, understand the effect of Notch signaling in other NE neoplasms, and study the effects of Notch signaling using Notch ligands.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Royal Thai Government Scholarship, Chulalongkorn University, Bangkok, Thailand (to V. S.), and NIH-National Cancer Institute Grants RO1CA70244 (to D. W. B.) and RO1CA47480 (to B. D. N.), and a training grant in Endocrinology and Metabolism T32 DK07751 (to D. R. A.). 
2 To whom requests for reprints should be addressed, at Johns Hopkins Oncology Center, 1650 Orleans Street, Room 553, Baltimore, MD 21231.
3 The abbreviations used are: HES, hairy enhancer of split; bHLH, basic helix-loop-helix; MASH1, mammalian achaete-scute homologue-1; NE, neuroendocrine; PNEC, pulmonary NE cell; hASH1, human achaete-scute homologue-1; SCLC, small cell lung cancer; CDKI, cyclin-dependent-kinase inhibitor; ERK, extracellular signal-regulated kinase; MEK, MAPK erk kinase; pMAPK, phosphorylated mitogen-activated-protein kinase; FBS, fetal bovine serum; GFP, green fluorescent protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly(ADP-ribose) polymerase; G3PDH, glyceraldehyde-3 phosphate dehydrogenase; Rb, retinoblastoma.
Received 9/28/00. Accepted 1/30/01.
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C. J. Strock, J.-I. Park, D. M. Rosen, B. Ruggeri, S. R. Denmeade, D. W. Ball, and B. D. Nelkin Activity of Irinotecan and the Tyrosine Kinase Inhibitor CEP-751 in Medullary Thyroid Cancer J. Clin. Endocrinol. Metab., January 1, 2006; 91(1): 79 - 84. [Abstract] [Full Text] [PDF]
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H. Osada, Y. Tatematsu, Y. Yatabe, Y. Horio, and T. Takahashi ASH1 Gene Is a Specific Therapeutic Target for Lung Cancers with Neuroendocrine Features Cancer Res., December 1, 2005; 65(23): 10680 - 10685. [Abstract] [Full Text] [PDF]
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 M. Kunnimalaiyaan, K. Traeger, and H. Chen Conservation of the Notch1 signaling pathway in gastrointestinal carcinoid cells Am J Physiol Gastrointest Liver Physiol, October 1, 2005; 289(4): G636 - G642. [Abstract] [Full Text] [PDF]
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 L. M. Sarmento, H. Huang, A. Limon, W. Gordon, J. Fernandes, M. J. Tavares, L. Miele, A. A. Cardoso, M. Classon, and N. Carlesso Notch1 modulates timing of G1-S progression by inducing SKP2 transcription and p27Kip1 degradation J. Exp. Med., July 5, 2005; 202(1): 157 - 168. [Abstract] [Full Text] [PDF]
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E. K. Nakakura, V. R. Sriuranpong, M. Kunnimalaiyaan, E. C. Hsiao, K. E. Schuebel, M. W. Borges, N. Jin, B. J. Collins, B. D. Nelkin, H. Chen, et al. Regulation of Neuroendocrine Differentiation in Gastrointestinal Carcinoid Tumor Cells by Notch Signaling J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 4350 - 4356. [Abstract] [Full Text] [PDF]
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V. Devgan, C. Mammucari, S. E. Millar, C. Brisken, and G. P. Dotto p21WAF1/Cip1 is a negative transcriptional regulator of Wnt4 expression downstream of Notch1 activation Genes & Dev., June 15, 2005; 19(12): 1485 - 1495. [Abstract] [Full Text] [PDF]
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K. Murata, M. Hattori, N. Hirai, Y. Shinozuka, H. Hirata, R. Kageyama, T. Sakai, and N. Minato Hes1 Directly Controls Cell Proliferation through the Transcriptional Repression of p27Kip1 Mol. Cell. Biol., May 15, 2005; 25(10): 4262 - 4271. [Abstract] [Full Text] [PDF]
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B. W. Purow, R. M. Haque, M. W. Noel, Q. Su, M. J. Burdick, J. Lee, T. Sundaresan, S. Pastorino, J. K. Park, I. Mikolaenko, et al. Expression of Notch-1 and Its Ligands, Delta-Like-1 and Jagged-1, Is Critical for Glioma Cell Survival and Proliferation Cancer Res., March 15, 2005; 65(6): 2353 - 2363. [Abstract] [Full Text] [PDF]
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 T. Shida, M. Furuya, T. Nikaido, T. Kishimoto, K. Koda, K. Oda, Y. Nakatani, M. Miyazaki, and H. Ishikura Aberrant Expression of Human Achaete-Scute Homologue Gene 1 in the Gastrointestinal Neuroendocrine Carcinomas Clin. Cancer Res., January 15, 2005; 11(2): 450 - 458. [Abstract] [Full Text] [PDF]
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J. B. Geigl, S. Langer, S. Barwisch, K. Pfleghaar, G. Lederer, and M. R. Speicher Analysis of Gene Expression Patterns and Chromosomal Changes Associated with Aging Cancer Res., December 1, 2004; 64(23): 8550 - 8557. [Abstract] [Full Text] [PDF]
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X. Fan, I. Mikolaenko, I. Elhassan, X. Ni, Y. Wang, D. Ball, D. J. Brat, A. Perry, and C. G. Eberhart Notch1 and Notch2 Have Opposite Effects on Embryonal Brain Tumor Growth Cancer Res., November 1, 2004; 64(21): 7787 - 7793. [Abstract] [Full Text] [PDF]
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H. Jafar-Nejad and H. J. Bellen Gfi/Pag-3/Senseless Zinc Finger Proteins: a Unifying Theme? Mol. Cell. Biol., October 15, 2004; 24(20): 8803 - 8812. [Full Text] [PDF]
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M. Noseda, L. Chang, G. McLean, J. E. Grim, B. E. Clurman, L. L. Smith, and A. Karsan Notch Activation Induces Endothelial Cell Cycle Arrest and Participates in Contact Inhibition: Role of p21Cip1 Repression Mol. Cell. Biol., October 15, 2004; 24(20): 8813 - 8822. [Abstract] [Full Text] [PDF]
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A. Kazanjian, D. Wallis, N. Au, R. Nigam, K. J. T. Venken, P. T. Cagle, B. F. Dickey, H. J. Bellen, C. B. Gilks, and H. L. Grimes Growth Factor Independence-1 Is Expressed in Primary Human Neuroendocrine Lung Carcinomas and Mediates the Differentiation of Murine Pulmonary Neuroendocrine Cells Cancer Res., October 1, 2004; 64(19): 6874 - 6882. [Abstract] [Full Text] [PDF]
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 F. Esni, B. Ghosh, A. V. Biankin, J. W. Lin, M. A. Albert, X. Yu, R. J. MacDonald, C. I. Civin, F. X. Real, M. A. Pack, et al. Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas Development, September 1, 2004; 131(17): 4213 - 4224. [Abstract] [Full Text] [PDF]
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 S.-L. Yu, H.-W. Chen, P.-C. Yang, K. Peck, M.-H. Tsai, J. J. W. Chen, and F.-Y. Lin Differential Gene Expression in Gram-negative and Gram-positive Sepsis Am. J. Respir. Crit. Care Med., May 15, 2004; 169(10): 1135 - 1143. [Abstract] [Full Text] [PDF]
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Y. Nefedova, P. Cheng, M. Alsina, W. S. Dalton, and D. I. Gabrilovich Involvement of Notch-1 signaling in bone marrow stroma-mediated de novo drug resistance of myeloma and other malignant lymphoid cell lines Blood, May 1, 2004; 103(9): 3503 - 3510. [Abstract] [Full Text] [PDF]
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M. Zayzafoon, S. A. Abdulkadir, and J. M. McDonald Notch Signaling and ERK Activation Are Important for the Osteomimetic Properties of Prostate Cancer Bone Metastatic Cell Lines J. Biol. Chem., January 30, 2004; 279(5): 3662 - 3670. [Abstract] [Full Text] [PDF]
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R. Qi, H. An, Y. Yu, M. Zhang, S. Liu, H. Xu, Z. Guo, T. Cheng, and X. Cao Notch1 Signaling Inhibits Growth of Human Hepatocellular Carcinoma through Induction of Cell Cycle Arrest and Apoptosis Cancer Res., December 1, 2003; 63(23): 8323 - 8329. [Abstract] [Full Text] [PDF]
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C. J. Strock, J.-I. Park, M. Rosen, C. Dionne, B. Ruggeri, S. Jones-Bolin, S. R. Denmeade, D. W. Ball, and B. D. Nelkin CEP-701 and CEP-751 Inhibit Constitutively Activated RET Tyrosine Kinase Activity and Block Medullary Thyroid Carcinoma Cell Growth Cancer Res., September 1, 2003; 63(17): 5559 - 5563. [Abstract] [Full Text] [PDF]
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V. Sriuranpong, J. I. Park, P. Amornphimoltham, V. Patel, B. D. Nelkin, and J. S. Gutkind Epidermal Growth Factor Receptor-independent Constitutive Activation of STAT3 in Head and Neck Squamous Cell Carcinoma Is Mediated by the Autocrine/Paracrine Stimulation of the Interleukin 6/gp130 Cytokine System Cancer Res., June 1, 2003; 63(11): 2948 - 2956. [Abstract] [Full Text] [PDF]
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F. H. Blackhall, M. Pintilie, M. Michael, N. Leighl, R. Feld, M.-S. Tsao, and F. A. Shepherd Expression and Prognostic Significance of Kit, Protein Kinase B, and Mitogen-activated Protein Kinase in Patients with Small Cell Lung Cancer Clin. Cancer Res., June 1, 2003; 9(6): 2241 - 2247. [Abstract] [Full Text] [PDF]
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D. Small, D. Kovalenko, R. Soldi, A. Mandinova, V. Kolev, R. Trifonova, C. Bagala, D. Kacer, C. Battelli, L. Liaw, et al. Notch Activation Suppresses Fibroblast Growth Factor-dependent Cellular Transformation J. Biol. Chem., April 25, 2003; 278(18): 16405 - 16413. [Abstract] [Full Text] [PDF]
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Z.-J. Liu, T. Shirakawa, Y. Li, A. Soma, M. Oka, G. P. Dotto, R. M. Fairman, O. C. Velazquez, and M. Herlyn Regulation of Notch1 and Dll4 by Vascular Endothelial Growth Factor in Arterial Endothelial Cells: Implications for Modulating Arteriogenesis and Angiogenesis Mol. Cell. Biol., January 1, 2003; 23(1): 14 - 25. [Abstract] [Full Text]
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B. Yan, N. Raben, and P. Plotz The Human Acid alpha -Glucosidase Gene Is a Novel Target of the Notch-1/Hes-1 Signaling Pathway J. Biol. Chem., August 9, 2002; 277(33): 29760 - 29764. [Abstract] [Full Text] [PDF]
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P. Muller, S. Kietz, J.-A. Gustafsson, and A. Strom The Anti-estrogenic Effect of All-trans-retinoic Acid on the Breast Cancer Cell Line MCF-7 Is Dependent on HES-1 Expression J. Biol. Chem., August 2, 2002; 277(32): 28376 - 28379. [Abstract] [Full Text] [PDF]
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