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Differential Notch1 and Notch2 expression and frequent activation of Notch signaling in gastric cancers.

Notch signaling plays pivotal roles in determining the manner of cell differentiation, tissue/organ formation, apoptosis in the early stages of development, and the processes of wound and healing of adult tissues. (1-4) So far, 4 members have been found in the Notch family, of which Notchl and Notch2 are well characterized genetically and functionally. (5,6) Recent data demonstrated that the biologic effects of Notch signaling varied in cell type-dependent fashions. (7,8) Moreover, Notchl and Notch2 proteins may function differently and even in reversed manners in the same cell types. These 2 proteins have different roles in the commitment and differentiation of neuronal and glial lineages of the olfactory epithelium during development. (9) In addition, Notchl rather than Notch2 is required for neuronal and glial differentiation in the cerebellum. (10) This evidence suggests that the signaling and functioning of Notch family proteins are not as simple as what has been supposed.

Cancer is the problem of mal-differentiation due to genetic and epigenetic alterations. (11-14) A body of evidence showed that Notch1 and Notch2 were involved in tumorigenic processes in the forms of altered upregulation or downregulation. (15,16) Because the biologic effects of individual Notch members may not be equivalent on different cancer types, contradictory results have been reported. For instance, Notch1 was regarded as a tumor suppressor, whereas Notch2 was regarded as a tumor promoter for medulloblastoma cells and B-cell chronic lymphocytic leukemia, (16,17) whereas a reverse situation was found in myeloma and breast cancers. (18,19) Furthermore, these 2 proteins work in a coordinated manner to promote cutaneous malignant melanoma (20) but to inhibit varicocele formation. (21) Apparently, a cautious evaluation should be performed on a cancer system in which the roles of the Notch pathway remain largely unknown.

Gastric cancer (GC) is one of the commonest malignancies in the world. The gastric epithelial cells undergo stepwise morphologic changes before malignant transformation. (22) Multiple signaling pathways have been known to be involved in gastrocarcinogenic processes. Concurrent activation of wingless-type MMTV integration site family member 2, signal transducer and activator of transcription 3, and nuclear factor of [kappa]B pathways were frequently found in the 2 major subtypes of GCs, intestinal GC (i-GC) and diffuse GC (d-GC), (23-25) which may have certain cross-talks with Notch signaling in the form of coactivation, (26) the molecular components shared by them, (27) and similar biologic consequence of their alterations to the cells. (28) However, the corresponding study of the Notch pathway in GCs has not yet been available. Our current study thus aimed to address this issue by checking the status of Notch signaling in GCs and their noncancerous and premalignant counterparts and by evaluating the influence of Notch inhibition in GC cells.

MATERIALS AND METHODS

Sample Collection

The detailed description including the human samples used and experimental protocols of the current study were reviewed by the institutional review board of Dalian Medical University, Dalian, China. After getting institutional review board approval and patients' consent, 74 GC surgical specimens and 10 endoscopic specimens were collected from the operating rooms at Dalian Medical University First and Second Affiliated Hospitals. The tissues were chosen from the tumor mass and, where possible, tissue surrounding the tumor, and then trimmed to suitable sizes on ice. After getting patients' consent, fresh endoscopic gastric biopsies from 10 GC-free patients were collected from the Gastroendoscopic Department of Dalian Medical University Second Affiliated Hospital. The samples were initially used for Helicobacter pylori testing and then for research purposes. Part of the tissue samples was snap-frozen immediately in liquid nitrogen and stored at--85[degrees]C until use; the remaining tissues were fixed with 10% formaldehyde for preparing paraffin blocks. Staining with hematoxylin-eosin was performed on each of the tissue blocks and a pathologic diagnosis determined before further experiments.

Tissue Microarray Construction and Immunohistochemical Staining

The representative donor tissues in tissue blocks were sampled with individual coring needles and orderly embedded into a recipient paraffin block according to the methods described elsewhere. (29) The tissue microarrays with densities of 56 spots/[cm.sup.2] were sectioned to 5-[micro]m thickness for immunohistochemical profiling of Notch1, Notch2, and Hes1 expression and intracellular distribution, respectively. The antibodies used were a mouse and a goat anti-human Notch1 antibody (1:100; MS-1339-P0, NeoMarkers, Inc, Fremont, California and 1:120; sc-6014, Santa Cruz Biotechnology, Inc, Santa Cruz, California), 2 rabbit anti-human Notch2 polyclonal antibodies (1:100; SC-5545, Santa Cruz Biotechnology and 1:200; ab8926, Abcam plc., Cambridge, United Kingdom), and a rabbit anti-human Hes1 polyclonal antibody (1:1000; a generous gift of Tetsuo Sudo, PhD, Toray Industries, Tokyo, Japan). The tissue microarray sections lacking incubation with individual primary antibodies were used as background controls. The staining results were evaluated by 2 researchers (H.L. and Y.S.), with the intensity of immunolabeling scored as negative (--), weakly positive (+), moderately positive (++), or strongly positive (+++).

Cell Culture and Analyses

Human GC cell lines AGS (30) and HGC-27 (31) were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia), and MGC803 (32) and BGC823 cell lines (33) were kindly provided by Beijing Cancer Institute, Peking University, Beijing, China. According to the literature, all of the 4 cell lines were established from poorly differentiated GCs. (30-33) The HGC-27, MGC803, and BGC823 cells were cultured in Dulbecco modified Eagle medium (Invitrogen Co, Grand Island, New York) containing 10% fetal bovine serum (Invitrogen) and AGS cells in Ham F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum at 37[degrees]C in 5% carbon dioxide. For immunocytochemical (ICC) and immunofluorescence (IF) studies, gelatincoated coverslips were put onto the dishes before initial cell seeding. When they grew to a semiconfluent state, the cell-bearing coverslips were collected for ICC and IF staining; the remaining cells were harvested for RNA and protein preparations, followed by Notch1-, Notch2-, and Hes1-oriented reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses.

RNA Isolation and RT-PCR

The cell pellets collected from each of the GC cell lines was placed immediately in a 1.5-mL Eppendorff tube containing 400-[micro]L ice-cold TriZol reagent (Invitrogen). RNA isolation was conducted according to the method described elsewhere. (34) RTPCR was performed using a Takara RNA PCR kit (Avian Myeloblastosis Virus version 2.1, Takara Inc Ltd, Dalian Branch, Dalian, China) under the following conditions: 0.8 mg of total RNA was reverse-transcribed at 55[degrees]C for 30 minutes in a 20-[micro]L solution containing random primer, and the reaction was terminated by incubating at 99[degrees]C for 5 minutes. [beta]-Actin, a quantitative and qualitative control, was polymerase chain reaction-amplified according to conditions described elsewhere (34) to generate a 326-bp fragment, followed by amplification of Notchl, Notchl, and Hesl transcripts with 3 pairs of specific polymerase chain reaction primers (Takara), respectively. The sequences of polymerase chain reaction primers for each of the gene transcripts are as follows: Notch1 (109 bp) upstream 5'-CCG CAG TTG TGC TCC TGA A-3', downstream 5'-ACC TTG GCG GTC TCG TAG CT-3' (16); Notch2 (533 bp) upstream 5'-TCG TGC AAG AGC CAG TTA CCC-3', downstream 5'-AAT GTC ATG GCC GCT TCA GAG-3' (35); Hes1 (254 bp) upstream 5'-CCA GTT TGC TTT CCT CAT TCC-3', downstream 5'-TCT TCT CTC CCA GTA TTC AAG TTC C-3'. Polymerase chain reaction products were separated on 1.5% agarose gels stained with 0.5 [micro]g/mL ethidium bromide and photographed with the automatic ultraviolet illuminator (UVP, Inc, Upland, California).

Protein Preparation and Western Blotting

The cell pellets were collected from the 4 GC cell lines, respectively. Each of them were mixed with 150-[micro]L prechilled 2 X Laemmli sodium dodecyl sulfate sample buffer containing 0.125M Tris-HCI, pH 6.8; 4% sodium dodecyl sulfate; 20% v/v glycerol; and 0.2M dithiothreitol. After being homogenized by ultrasonic vibration (UP200s; Hielscher Ultrasonics GmbH, Teltow, Germany), the samples were heated in boiling water for 5 minutes. The protein-containing supernatant liquids were removed to fresh tubes for experimental purposes.

For Western blot analysis, the protein samples (100 [micro]g/sample) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Amersham Biosciences Inc, Bucks, United Kingdom). The prepared membrane was blocked with 5% skim milk (Becton Dickinson France SAS, le Pont de Claix, France) in Tris-buffered saline Tween-20 at 4[degrees]C overnight. The membrane was then incubated at room temperature for 1 hour with anti-Notch1 antibody (C-20; sc-6014), followed by 3 washes in blocking solution for 10 minutes each. Secondary antibody incubation was conducted for 1 hour with horseradish peroxidase-conjugated rabbit anti-mouse IgG (1:3000). Bound antibody was detected using the enhanced chemiluminescence solution (Roche Diagnostic GmbH, Mannheim, Germany) and exposing the membrane to x-ray film (Kodak Co, Rochester, New York). After removing the labeling signal by incubation with stripping buffer (62.5mM Tris-HCI, pH 6.7; 100mM 2-Mercaptoetho nal; 2% sodium dodecyl sulfate) at 55[degrees]C for 30 minutes, the membrane was reprobed with anti-Notch2 polyclonal antibody (ab8926) and thereafter with a rabbit anti-human Hes1 polyclonal antibody.

Treatments With [gamma]-Secretase Inhibitors

To elucidate the importance of Notch1 and Notch2 in the growth of GC cells, Notch2-expressing AGS and BGC823 cell lines were treated by 2 [gamma]-secretase inhibitors, respectively. The experiment was set in 4 groups as group 1, normal culture; group 2, treatment with 8[micro]M L-685,458 ([gamma]-secretase inhibitor X; EMD Biosciences, Inc, La Jolla, California); group 3, treatment with 10[micro]M N-[N-(3,5-difluorophenacetyl)-L-ananyl]-S-phenyglycine t-butyl ester ([gamma]-secretase inhibitor IX/DAPT; EMD Biosciences) (36,37); and group 4, treatment with 0.2% dimethyl sulfoxide, the organic solvent for L-685,458 and DAPT. Total cell numbers and cell viability were determined in 12-hour intervals by staining the suspended single cells with 0.25% trypan blue. (38) The stained and unstained cells were counted by 3 individuals with hemocytometers. Meanwhile, the cell-bearing coverslips were collected at 4-hour intervals and fixed in cold acetone for Hes1-oriented ICC staining and IF. The treatments lasted for 48 hours and were repeated at least 3 times.

Statistical Analyses

Mann-Whitney tests were used to analyze the status of Notch1, Notch2, and Hes1 expression in different histologic groups. The Spearman correlation test was used to determine the correlation of Hes1 expression with Notch1 expression and Notch2 expression and Notch1, Notch2, and Hes1 expression with malignant transformation. The level of statistical significance was always set at P < .05. All P values are 2-sided. Statistical analysis was performed using the statistical software SPSS version 11.5 (SPSS, Inc, Chicago, Illinois).

RESULTS

Histologic Examination and Classification

Gastric cancer tissue and, when possible, the premalignant mucosa surrounding the tumor, were taken from the same surgical specimen during sample collection. Histologic reevaluation was performed on them prior to further experiments. A total of 42 GC surrounding premalignant tissues (24 chronic gastritis and 18 intestinal metaplasia [IM]) and 74 cancer tissues (48 i-GC and 26 d-GC) were examined in this study. Ten gastroendoscopic biopsies with mild inflammatory changes were obtained from GC-free patients and used as relatively normal controls.

Preferable Notch1 Expression and Nuclear Translocation in IM and i-GCs

The results of tissue microarray-based immunohistochemical staining are summarized in Table 1 and shown in Figure 1, A through E and Figure 2. It was found that Notch1 expression and nuclear translocation was absent in all of the 10 noncancerous endoscopic gastric mucosa specimens and became detectable in 20.8% (5 of 24) and 16.7% (4 of 24) of chronic gastritis and in 61.1% (11 of 18) and 50.0% (9 of 18) of IMs, respectively. The rates of Notch1 detection were 58.3% (28 of 48) and 54.2% (26 of 48) in i-GCs and the same 23.1% (6 of 26) in d-GCs of cytosolic and nuclear stainings, respectively. Statistical analyses revealed that the frequencies of Notch1 expression and nuclear translocation were significantly different between (1) the noncancerous group and premalignant (cytoplasm, P = .02; nuclear, P = .046) or GC group (cytoplasm, P = .007; nuclear, P = .01), (2) the chronic gastritis and IM tissues (cytoplasm, P = .009; nuclear, P = .03), and (3) the i-GC and d-GC group (cytoplasm, P = .002; nuclear, P = .005). In comparison with the results of normal gastric tissues, the incidence of Notch1 detection was distinctly increased in IM and i-GC (cytoplasm, r = 0.236, P = .04; nuclear, r = 0.290, P = .01) but not in chronic gastritis and d-GC (cytoplasm, r = 0.160, P = .22; nuclear, r = 0.191, P = .14).

[FIGURE 1 OMITTED]

Correlation of Notch2 Nuclear Translocation With GC Formation

As shown in Table 1, Figure 1, F through J, and Figure 2, Notch2 was expressed in 20.0% (2 of 10) of noncancerous endoscopic gastric mucosa, and only 1 (10.0%) was found with nuclear translocation; in contrast, 73.8% (31 of 42) of premalignant lesions were positive for Notch2 expression and 71.4% (30 of 42) with nuclear staining, showing a significant difference between these 2 groups in expression and nuclear translocation (cytoplasm, P = .002; nuclear, P = .001). The tendency of Notch2 upregulation became more distinct among GC tissues in increased rates and levels of expression (98.6%; 73 of 74) and nuclear translocation (97.3%; 72 of 74; Table 1). Statistical analysis revealed that the frequency of Notch2 expression and nuclear translocation in the GC group was significantly different from that of noncancerous (cytoplasm, P < .001; nuclear, P < .001) and premalignant samples (cytoplasm, P < .001; nuclear, P < .001), whereas no statistical significance of Notch2 expression was found between i-GC and d-GC (cytoplasm, P = .39; nuclear, P = .38). Correlative study showed that Notch2 expression (r = 0.474, P < .001) and nuclear translocation (r = 0.585, P < .001) were closely linked with both i-GC and d-GC formation (P < .001).

Close Correlation of Hes1 and Notch2 Nuclear Translocation

The rates of nuclear Hes1 detection were 10.0% (1 of 10) in noncancerous endoscopic epithelial tissues, 42.9% (18 of 42) in premalignant lesions, and 81.1% (60 of 74) in GC cases (Table 1; Figure 1, K through O), which were significantly different between GCs and the premalignant lesions (P < .001) or the noncancerous mucosa (P < .001). Mann-Whitney tests revealed distinct differences between noncancerous mucosa and premalignant lesions (P = .04) but not between i-GCs and d-GCs (P = .59). Correlative study showed a close relation of Hes1 nuclear translocation with malignant transformation of gastric epithelia (r = 0.474, P < .001). Activation of Notch signaling was decided when Hes1 appeared in the nuclei accompanied with Notch1 and/or Notch2 nuclear translocation (Figure 2). As showed in Table 2, nuclear labeling of Hes1 was correlated with Notch2 (r = 0.442, P < .001) rather than Notch1 (r = 0.072, P = .42) expression.

Constitutive Hes1 and Notch2 Expression in GC Cell Lines

Four established human GC cell lines were used to validate the previously discussed in vivo findings, which had been derived from poorly differentiated adenocarcinomas. (30-33) They were checked with the same parameters for tissue samples by ICC and IF staining combined with RT-PCR and Western blot analyses. Both ICC and IF demonstrated that Notch2 and Hes1 were detected in BGC823, MGC803, AGS, and HGC-27 cells, whereas none of those cell lines was found with Notch1 expression (Figure 3, A through C). Consistent with the previously mentioned findings, RT-PCR generated sufficient amounts of Notch2 and Hes1, whereas Notch1 was almost undetectable in the 4 cell lines (Figure 3, D). Accordingly, Western blot analyses clearly demonstrated a 100-kDa Notch2 band and a 45-kDa Hes1 band but no sign of Notch1 production in the 4 cell lines (Figure 3, D).

[FIGURE 2 OMITTED]

[gamma]-Secretase Inhibitors Suppressed Hes1 Expression But Not Cell Proliferation

To evaluate potential impact(s) of Notch activation in GC cells, 2 specific inhibitors of g-secretase, L-685,458 ([gamma]-secretase inhibitor X) and DAPT, were used to inhibit proteolytic activation of Notch signaling in AGS and BGC823 cells, respectively. The results revealed that after 8[micro]M L-685,458 treatment for 36 hours or 10[micro]M DAPT treatment for 48 hours, Notch activation was blocked efficiently in reduced cytoplasmic distribution and almost diminished nuclear translocation of Hes1 proteins in both AGS (Figure 4, A through C) and BGC823 cells (data not shown). However, these treatments neither brought about growth arrest nor cell death (Figure 4, D).

COMMENT

Notch signaling is activated dynamically during evolution and plays active roles in cell fate determination and differentiation during embryonic development. Therefore, alteration of this signaling may lead to some abnormalities such as cancers (39-41) and developmental disorders. (42) Multiple Notch family members have been identified, of which Notch1 and Notch2 are well-categorized. Extensive investigations revealed that these 2 Notch proteins functioned in tumor type-related fashion and their roles may not be necessarily identical in different kinds of cancers. (16-21) The multifaceted features of Notch family members suggest the necessity to check the activation pattern(s) and potential roles of this signaling in different tumor types without any initial impression.

The biologic functions of Notch1 vary greatly with cancer types. For example, Notch1 was regarded as a target gene of p53 and involved in human keratinocyte tumor suppression, (43) whereas it was proved to directly regulate c-Myc gene transcription and promote the growth of leukemia and mammary cancer cells. (44-46) More recent study revealed that activated Notch signaling has little influence in the growth and survival of medulloblastoma cells. (47) According to the results of the current study, Notch1 was rarely expressed in relatively normal gastric mucosa and appeared in premalignant and cancer tissues. Interestingly, Notch1 was found to be more preferably expressed with distinct nuclear translocation in IM and well-differentiated i-GC, whereas the 4 GC cell lines derived from poorly or undifferentiated GCs were negative in its expression. Because Notch signaling is important in regulating the regeneration, differentiation, and proliferation of the intestinal epithelial cells, (48) we therefore speculate that Notch1 may play certain role(s) in promoting metaplastic transition of gastric epithelial cells and/or in maintaining constant proliferation of intestinalized epithelial cells.

It has been reported that Notchl acts as an oncogene in some types of cancers but its status in GCs remains obscure. It was found in this study that Notch2 expression and nuclear translocation was uncommon in endoscopic noncancerous mucosa with mild inflammatory changes and became frequent in both i-GC (98.6%) and d-GC (97.3%) cases. Western blot analysis demonstrated that the original and proteolytic forms of Notch2 were detected in all of the 4 GC cell lines, suggesting the close correlation of Notch2 expression and proteolytic activation with stepwise gastrocarcinogenesis. Hesl is a famous target gene of Notch signaling (49) because its expression can be triggered when the fragments of cleaved Notch intracellular domain translocalize to nucleus, associate with the DNA-binding-protein, and convert the latter to Hes1 activator. (6) Therefore, Hes1 expression and nuclear ending have been regarded as the indicator of Notch activation. (50) Unlike the situation of Notch1, coexpression and, more importantly, the nuclear cotranslocation of Notch2 and Hes1 were more frequent in vivo and in vitro. Because all the d-GC cases so far checked are positive in Notch2 and only 23.1% of them are positive in Notch1, it is reasonable to consider that Notch2-mediated signaling would be more important for GC formation and progression.

Recent findings have revealed that Notch signaling can variously promote or suppress tumor growth through regulating differentiation, cell cycle progression, angiogenesis, and possibly cancer stem cell self-renewal and immune function. (7,51,52) Nevertheless, the knowledge about the functional aspect of Notch in gastrocarcinogenesis is still limited. Given the evidence of prominent Notch-associated Hes1 expression in GCs and their related lesions, the importance of Notch signaling in the growth and survival of Notch2-activating GC cells was evaluated by inhibiting proteolytic cleavage of Notch transmembrane protein with 2 [gamma]-secretase antagonists. Although Hes1 expression and nuclear translocation were inhibited efficiently by the 2 reagents, both cell lines showed neither growth arrest nor cell death. These phenomena suggest that Notch2 signaling may not be the critical molecular machinery for the maintenance and survival of GC cells. Multiple signaling pathways are activated during gastrocarcinogenesis, of which some such as wingless-type MMTV integration site family member and nuclear factor of [kappa]B signaling have certain functional cross-links with Notch signaling. (53-56) Therefore, it would be possible that other signaling pathways may compensate Notch signaling when its biologic activity is suppressed. In this context, simply blocking Notch signaling may not achieve promising inhibitory outcome in a GC experimental system.

Taken together, the Notch1 and Notch2 expression patterns and the status of Notch signaling during stepwise gastrocarcinogenesis were profiled in this study, which revealed a close correlation of Notch activation with GC formation. Because blockage of Notch proteolytic activation failed to inhibit growth and to induce cell death of GC cells, it would be possible that beyond Notch signaling, other more powerful cell maintaining systems may be present in GCs. However, the favorable Notch1 expression in gastric lesions with intestinal phenotypes and the frequent Notch2 and Hes1 expression in different premalignant and cancer tissues can still be regarded as adverse biologic indicators of GC risk. As the next research step, it would be worthwhile to check the expression and functional aspect(s) of Notch1 in i-GC cell lines and to elucidate whether inhibition of all Notch signaling could offset the effect of either Notch1 or Notch2 alone on proliferation and survival of GC cells.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

This work is supported in part by grants from the National Natural Science Foundation of China (30527002, 30670946, 30700365, and 81071971). We thank the surgeons and nurses in the Department of General Surgery, Dalian Medical University First and Second Affiliated Hospitals for providing gastric specimens; the doctors in the Department of Pathology, Dalian Medical University First Affiliated Hospital for pathologic consultation; and Qi-Gui Liu, PhD, in the Department of Medical Statistics, Dalian Medical University for his help with statistical analyses. We thank Tetsuo Sudo, PhD, from Toray Industries, Tokyo, Japan for providing us with anti-Hes1 antibody.

References

(1.) Ohishi K, Katayama N, Shiku H, et al. Notch signaling in hematopoiesis. Semin Cell Dev Biol. 2003;14(2):143-150.

(2.) Yang X, Klein R, Tian X, et al. Notch activation induces apoptosis in neural progenitor cells through a p53-dependent pathway. Dev Biol. 2004;269(1):8194.

(3.) Nicolas M, Wolfer A, Raj K, et al. Notch1 functions as a tumor suppressor in mouse skin. Nat Genet. 2003;33(3):416-421.

(4.) Adolphe C, Wainwright B. Pathways to improving skin regeneration. Expert Rev Mol Med. 2005;7(20):1-14.

(5.) Hansson EM, Lendahl U, Chapman G. Notch signaling in development and disease. Semin Cancer Biol. 2004;14(5):320-328.

(6.) Mumm JS, Kopan R. Notch signaling: from the outside in. Dev Biol. 2000; 228(2):151-165.

(7.) Roy M, Pear WS, Aster JC. The multifaceted role of Notch in cancer. Curr Opin Genet Dev. 2007;17(1):52-59.

(8.) Bray SJ. Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol. 2006;7(9):678-689.

(9.) Carson C, Murdoch B, Roskams AJ. Notch 2 and Notch 1/3 segregate to neuronal and glial lineages of the developing olfactory epithelium. Dev Dyn. 2006;235(6):1678-1688.

(10.) Lutolf S, Radtke F, Aguet M, et al. Notch1 is required for neuronal and glial differentiation in the cerebellum. Development. 2002;129(2):373-385.

(11.) Takata M, Lin J, Takayanagi S, et al. Genetic and epigenetic alterations in the differential diagnosis of malignant melanoma and spitzoid lesion. Br J Dermatol. 2007;156(6):1287-1294.

(12.) Song J, Kim CJ, Cho YG, et al. Genetic and epigenetic alterations of the KLF6 gene in hepatocellular carcinoma. J Gastroenterol Hepatol. 2006;21(8): 1286-1289.

(13.) Wolff EM, Liang G, Jones PA. Mechanisms of disease: genetic and epigenetic alterations that drive bladder cancer. Nat Clin Pract Urol. 2005;2(10): 502-510.

(14.) Tsigris C, Chatzitheofylaktou A, Xiromeritis C, et al. Genetic association studies in digestive system malignancies. Anticancer Res. 2007;27(5B):3577 3587.

(15.) Katoh M. Networking of WNT, FGF, Notch, BMP, and Hedgehog signaling pathways during carcinogenesis. Stem Cell Rev. 2007;3(1):30-38.

(16.) Fan X, Mikolaenko I, Elhassan I, et al. Notch1 and notch2 have opposite effects on embryonal brain tumor growth. Cancer Res. 2004;64(21):7787-7793.

(17.) Hubmann R, Schwarzmeier JD, Shehata M, et al. Notch2 is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia. Blood. 2002; 99(10):3742-3747.

(18.) Nefedova Y, Cheng P, Alsina M, et al. Involvement of Notch-1 signaling in bone marrow stroma-mediated de novo drug resistance of myeloma and other malignant lymphoid cell lines. Blood. 2004;103(9):3503-3510.

(19.) Parr C, Watkins G, Jiang WG. The possible correlation of Notch1 and Notch2 with clinical outcome and tumour clinicopathological parameters in human breast cancer. Int J Mol Med. 2004;14(5):779-786.

(20.) Massi D, Tarantini F, Franchi A, et al. Evidence for differential expression of Notch receptors and their ligands in melanocytic nevi and cutaneous malignant melanoma. Mod Pathol. 2006;19(2):246-254.

(21.) Sahin Z, Bayram Z, Celik-Ozenci C, et al. Effect of experimental varicocele on the expressions of Notch 1, 2, and 3 in rat testes: an immunohistochemical study. Fertil Steril. 2005;83(1):86-94.

(22.) Moss SF, Malfertheiner P. Helicobacter and gastric malignancies. Helicobacter. 2007;12(suppl 1):23-30.

(23.) Cheng XX, Wang ZC, Chen XY, et al. Correlation of Wnt-2 expression and beta-catenin intracellular accumulation in Chinese gastric cancers: relevance with tumour dissemination. Cancer Lett. 2005;223(2):339-347.

(24.) Cheng XX, Wang ZC, Chen XY, et al. Frequent loss of membranous E-cadherin in gastric cancers: a cross-talk with Wnt in determining the fate of beta catenin. Clin Exp Metastasis. 2005;22(1):85-93.

(25.) Han JC, Zhang KL, Chen XY, et al. Expression of seven gastric cancer-associated genes and its relevance for Wnt, NF-kappaB and Stat3 signaling. APMIS. 2007;115(12):1331-1343.

(26.) Espinosa L, Ingles-Esteve J, Aguilera C, et al. Phosphorylation by glycogen synthase kinase-3 beta down-regulates Notch activity, a link for Notch and Wnt pathways. J Biol Chem. 2003;278(34):32227-32235.

(27.) Balaraman Y, Limaye AR, Levey AI, et al. Glycogen synthasekinase 3beta and Alzheimer's disease: pathophysiological and therapeutic significance. Cell Mol Life Sci. 2006;63(11):1226-1235.

(28.) Katoh M. Dysregulation of stem cell signaling network due to germline mutation, SNP, Helicobacter pylori infection, epigenetic change and genetic alteration in gastric cancer. Cancer Biol Ther. 2007;6(6):832-839.

(29.) Li H, Sun Y, Kong QY, et al. Combination of nucleic acid and protein isolation with tissue array construction: using defined histologic regions in single frozen tissue blocks for multiple research purposes. Int J Mol Med. 2003;12(3): 299-304.

(30.) Moon WS, Tarnawski AS, Chai J, et al. Reduced expression of epidermal growth factor receptor related protein in gastric cancer. Gut. 2005;54(2):201-206.

(31.) Akagi T, Kimoto T. Human cell line (HGC-27) derived from the metastatic lymph node of gastric cancer. Acta Med Okayama. 1976;30(3):215-219.

(32.) Zhang WG, Ma LP, Wang SW, et al. Antisense bcl-2 retrovirus vector increases the sensitivity of a human gastric adenocarcinoma cell line to photodynamic therapy. Photochem Photobiol. 1999;69(5):582-586.

(33.) Deng GR, Lu YY, Chen SM, et al. Activated c-Ha-ras oncogene with a guanine to thymine transversion at the twelfth codon in a human stomach cancer cell line. Cancer Res. 1987;47(12):3195-3198.

(34.) Chen XY, Wang ZC, Li H, et al. Nuclear translocations of beta-catenin and TCF4 in gastric cancers correlate with lymph node metastasis but probably not with CD44 expression. Hum Pathol. 2005;36(12):1294-1301.

(35.) Walsh J, Andrews PW. Expression of Wnt and Notch pathway genes in a pluripotent human embryonal carcinoma cell line and embryonic stem cell. APMIS. 2003;111(1):197-210.

(36.) Morohashi Y, Kan T, Tominari Y, et al. C-terminal fragment of presenilin is the molecular target of a dipeptidic gamma-secretase-specific inhibitor DAPT(N[N-(3,5-difluorophenacetyl)-L-alanyl]-S- phenylglycine t-butyl ester). J Biol Chem. 2006;281(21):14670-14676.

(37.) Hallahan AR, Pritchard JI, Hansen S, et al. The SmoA1 mouse model reveals that notch signaling is critical for the growth and survival of sonic hedgehog-induced medulloblastomas. Cancer Res. 2004;64(21):7794-7800.

(38.) Robert-Moreno A, Espinosa L, Sanchez MJ, et al. The notch pathway positively regulates programmed cell death during erythroid differentiation. Leukemia. 2007;21(7):1496-1503.

(39.) Rusten TE, Haglund K, Stenmark H. Aberrant receptor signaling and trafficking as mechanisms in oncogenesis. Crit Rev Oncol. 2007;13(1): 39-74.

(40.) Yao J, Duan L, Fan M, et al. gamma-secretase inhibitors exerts antitumor activity via down-regulation of Notch and Nuclear factor kappa B in human tongue carcinoma cells. Oral Dis. 2007;13(6):555-563.

(41.) Wuest M, Dummer R, Urosevic M. Induction of the members of Notch pathway in superficial basal cell carcinomas treated with imiquimod. Arch Dermatol Res. 2007;299(10):493-498.

(42.) Fan X, Matsui W, Khaki L, et al. Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors. Cancer Res. 2006; 66(15):7445-7452.

(43.) Lefort K, Mandinova A, Ostano P, et al. Notch1 is a p53 target gene involved in human keratinocyte tumor suppression through negative regulation of ROCK1/2 and MRCKalpha kinases. Genes Dev. 2007;21(5):562-577.

(44.) Weng AP, Millholland JM, Yashiro-Ohtani Y, et al. c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma. Genes Dev. 2006;20(15):2096-2109.

(45.) Sharma VM, Draheim KM, Kelliher MA. The Notch1/c-Myc pathway in T cell leukemia. Cell Cycle. 2007;6(8):927-930.

(46.) Klinakis A, Szabolcs M, Politi K, et al. Myc is a Notch1 transcriptional target and a requisite for Notch1-induced mammary tumorigenesis in mice. Proc Natl Acad Sci U S A. 2006;103(24):9262-9267.

(47.) Wang Q, Li H, Liu N, et al. Correlative analyses of notch signaling with resveratrol-induced differentiation and apoptosis of human medulloblastoma cells. Neurosci Lett. 2008;438(2):168-173.

(48.) Okamoto R, Tsuchiya K, Nemoto Y, et al. Requirement of Notch activation during regeneration of the intestinal epithelia. Am J Physiol Gastrointest Liver Physiol. 2009;296(1):G23-G35.

(49.) Ross DA, Rao PK, Kadesch T. Dualroles for the Notch target gene Hes1 in the differentiation of 3T3-L1 preadipocytes. Mol Cell Biol. 2004;24(8):3505-3513.

(50.) Chiaramonte R, Calzavara E, Balordi F, et al. Differential regulation of Notch signal transduction in leukaemia and lymphoma cells in culture. J Cell Biochem. 2003;88(3):569-577.

(51.) Kageyama R, Ohtsuka T, Tomita K. The bHLH gene Hes1 regulates differentiation of multiple cell types. Mol Cells. 2000;10(1):1-7.

(52.) Miele L, Golde T, Osborne B. Notch signaling in cancer. Curr Mol Med. 2006;6(8):905-918.

(53.) Das AV, Bhattacharya S, Zhao X, et al. The canonical Wnt pathway regulates retinal stem cells/progenitors in concert with Notch signaling. Dev Neurosci. 2008;30(6):389-409.

(54.) Shimizu T, Kagawa T, Inoue T, et al. Stabilized beta-catenin functions through TCF/LEF proteins and the Notch/RBP-Jkappa complex to promote proliferation and suppress differentiation of neural precursor cells. Mol Cell Biol. 2008;28(24):7427-7441.

(55.) Fukushima H, Nakao A, Okamoto F, et al. The association of Notch2 and NF-kappaB accelerates RANKL-induced osteoclastogenesis. Mol Cell Biol. 2008; 28(20):6402-6412.

(56.) Osipo C, Golde TE, Osborne BA, et al. Off the beaten pathway: the complex cross talk between Notch and NF-kappaB. Lab Invest. 2008;88(1):11-17.

Yuan Sun, MD; Xue Gao, MD;Jia Liu, MD, PhD; Qing-You Kong, BS; Xiao-Wei Wang, MS; Xiao-Yan Chen, BS; Qian Wang, PhD; Yi-Fan Cheng, MS; Xiao-Xia Qu, MS; Hong Li, MD, PhD

Accepted for publication June 14, 2010.

From the Liaoning Laboratory of Cancer Genomics and Department of Cell Biology (Drs Sun, Gao, Liu, Q. Wang, and Li, Mr Kong, and Mss Xiao-W. Wang and Chen) and the Faculty of Clinical Medicine, College of Basic Medical Sciences (Mss Cheng and Qu), Dalian Medical University, Dalian, China; and the Department of Pathology, the First Affiliated Hospital of Dalian Medical University, Dalian, China (Dr Gao).

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Hong Li, MD, PhD, Liaoning Laboratory of Cancer Genomics, Dalian Medical University, Dalian 116044, China (e-mail: lihongmcn@yahoo.com.cn).
Table 1. Intracellular Distribution of Notch1, Notch2, and Hes1 in
Different Gastric Histologic Groups

 Notch1
Notch2

 Cytoplasm, No. (%)/Nuclear, No. (%)

Histology No. - +

Noncancerous 10 10 (100)/10 (100) 0 (0)/0 (0)
Premalignant 42 26 (62)/29 (69) 12 (29)/7 (17)
Chronic gastritis 24 19 (79)/20 (83) 4 (17)/2 (8)
Intestinal metaplasia 18 7 (39)/9 (50) 8 (44)/5 (28)
Gastric cancer 74 40 (54)/42 (57) 28 (38)/13 (18)
Intestinal type 48 20 (42)/22 (46) 22 (46)/9 (19)
Diffuse type 26 20 (77)/20 (77) 6 (23)/4 (15)

 Notch1

 Cytoplasm, No. (%)/Nuclear, No. (%)

Histology No. ++ +++

Noncancerous 10 0 (0)/0 (0) 0 (0)/0 (0)
Premalignant 42 4 (10)/6 (14) 0 (0)/0 (0)
Chronic gastritis 24 1 (4)/2 (8) 0 (0)/0 (0)
Intestinal metaplasia 18 3 (17)/4 (22) 0 (0)/0 (0)
Gastric cancer 74 5 (7)/18 (24) 1 (1)/1 (1)
Intestinal type 48 5 (10)/16 (33) 1 (2)/1 (2)
Diffuse type 26 0 (0)/2 (8) 0 (0)/0 (0)

 Notch1 Notch2

 Cytoplasm, No. (%)/Nuclear, No. (%)

Histology No. P (a) -

Noncancerous 10 8 (80)/9 (90)
Premalignant 42 ([dagger]) 11 (26)/12 (29)
Chronic gastritis 24 ([double dagger]) 10 (42)/11 (46)
Intestinal metaplasia 18 1 (6)/1 (6)
Gastric cancer 74 ([dagger]) 1 (1)/2 (3)
Intestinal type 48 ([section]) 1 (2)/2 (4)
Diffuse type 26 0 (0)/0 (0)

 Notch2

 Cytoplasm, No. (%)/Nuclear, No. (%)

Histology No. + ++

Noncancerous 10 2 (20)/1 (10) 0 (0)/0 (0)
Premalignant 42 21 (50)/10 (24) 8 (19)/19 (45)
Chronic gastritis 24 10 (42)/5 (21) 4 (17)/8 (33)
Intestinal metaplasia 18 11 (61)/5 (28) 4 (22)/11 (61)
Gastric cancer 74 37 (50)/12 (16) 30 (41)/33 (45)
Intestinal type 48 22 (46)/7 (15) 20 (42)/19 (40)
Diffuse type 26 15 (58)/5 (19) 10 (38)/14 (54)

 Notch2

 Cytoplasm, No. (%)/Nuclear, No. (%)

 Histology No. +++ P (a)

Noncancerous 10 0 (0)/0 (0)
Premalignant 42 2 (5)/1 (2) ([dagger])
Chronic gastritis 24 0 (0)/0 (0) ([double dagger])
Intestinal metaplasia 18 2 (11)/1 (6)
Gastric cancer 74 6 (8)/27 (36) ([dagger]) *
Intestinal type 48 5 (10)/20 (42)
Diffuse type 26 1 (4)/7 (27)

 Hes1

 Nuclear, No. (%)

Histology No. - + ++ +++

Noncancerous 10 9 (90) 1 (10) 0 (0) 0 (0)
Premalignant 42 24 (57) 6 (14) 12 (29) 0 (0)
Chronic gastritis 24 17 (71) 3 (12) 4 (17) 0 (0)
Intestinal metaplasia 18 7 (39) 3 (17) 8 (44) 0 (0)
Gastric cancer 74 14 (19) 19 (26) 27 (36) 14 (19)
Intestinal type 48 8 (17) 13 (27) 17 (35) 10 (21)
Diffuse type 26 6 (23) 6 (23) 10 (38) 4 (15)

 Hes1

Histology No. P (a)

Noncancerous 10
Premalignant 42 ([dagger])
Chronic gastritis 24 ([double dagger])
Intestinal metaplasia 18
Gastric cancer 74 ([dagger]) *
Intestinal type 48
Diffuse type 26

Abbreviations: -, negative for immunolabeling in target cells; +,
weakly positive for immunolabeling; ++, moderately positive for
immunolabeling; +++, highly positive for immunolabeling.

(a) Established statistical difference P < .05 in comparison with
premalignant lesions *, with noncancerous group ([dagger]), with
intestinal metaplasia ([double dagger]), and with diffuse gastric
cancer ([section]).

Table 2. Correlation of Hes1 Expression With Notch1 and Notch2
Nuclear Translocation

 Notch1

Hes1 n - + ++ +++ r P

- 47 33 8 6 0 0.072 .42
+ 26 14 4 7 1
++ 39 26 5 8 0
+++ 14 8 3 3 0

 Notch2

Hes1 - + ++ +++ r P

- 19 7 18 3 0.442 <.001
+ 2 7 11 6
++ 2 8 17 12
+++ 0 1 6 7

Abbreviations: -, negative for immunolabeling for target cells; +,
weakly positive for immunolabeling; ++, moderately positive for
immunolabeling; +++, highly positive for immunolabeling.
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Author:Sun, Yuan; Gao, Xue; Liu, Jia; Kong, Qing-You; Wang, Xiao-Wei; Chen, Xiao-Yan; Wang, Qian; Cheng, Yi
Publication:Archives of Pathology & Laboratory Medicine
Date:Apr 1, 2011
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