Quantitative analysis of colorectal tissue microarrays by immunofluorescence and <i>in situ</i> hybridization

Jubb, A. M.; Landon, TH; Burwick, Jennifer A.; Pham, TQ; Frantz, GD; Cairns, Brian E.; Quirke, Philip; Peale, F V; Hillan, K J

doi:10.1002/path.1371

Cited by 41 publications

(33 citation statements)

References 51 publications

(72 reference statements)

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“…An additional eight unmatched hepatic metastases from nine patients were also included. TMAs were constructed from triplicate tissue cores (0.6 mm in diameter) as described previously (Jubb et al, 2003). A 'normal' TMA represented three samples of each: aorta, bladder, brain, breast, colon, heart, kidney, lung, lymph node, ovary, pancreas, placenta, prostate, seminal vesicles, skin, small intestine, spleen, stomach, testis, thyroid and tonsil.…”

Section: Oligonucleotide Microarraysmentioning

confidence: 99%

“…Primers sequences are provided in Supplementary Table 3. Isotopic probe labelling, hybridization, washing and developing have been described elsewhere (Jubb et al, 2003). Tissues subjected to in situ hybridization for b-actin and ascl2 were scored positive or negative by a single pathologist for the presence or absence of the hybridized antisense probe, throughout the tissue (b-actin) or in >10% of the epithelium (ascl2).…”

Section: Oligonucleotide Microarraysmentioning

confidence: 99%

“…Immunohistochemistry was performed as described elsewhere (Jubb et al, 2003) with a mouse anti-b-catenin monoclonal antibody (clone 14, BD Pharmingen, San Diego, CA, USA) or naive immunoglobulins at 1 mg ml À1 . In all TMAs, the score from the highest expressing core was used to represent each patient.…”

Section: Immunohistochemistrymentioning

confidence: 99%

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Achaete-scute like 2 (ascl2) is a target of Wnt signalling and is upregulated in intestinal neoplasia

Jubb

Chalasani²,

Frantz³

et al. 2006

Oncogene

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121

112

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Achaete-scute like (ASCL)2 is a basic helix-loop-helix transcription factor essential for the maintenance of proliferating trophoblasts during placental development. Using oligonucleotide microarrays we identified ascl2 as a gene significantly upregulated in colorectal adenocarcinomas (n ¼ 36 cancers, n ¼ 16 normals; 15-fold, Po0.0001). This finding was confirmed by quantitative reverse transcriptase (RT)-PCR on large intestinal cancers (n ¼ 29 cancers, n ¼ 16 normals; 10-fold, Po0.0001).In situ hybridization for ascl2 demonstrated expression at the base of small and large intestinal crypts (n ¼ 304), but in no other normal tissues excepting placenta. By in situ hybridization, 52-71% of colorectal adenomas (n ¼ 187), 50-73% of large (n ¼ 327) and 33-64% of small intestinal adenocarcinomas (n ¼ 124) were positive for ascl2 expression. Upregulation of murine ascl2 was also observed using oligonucleotide microarrays, quantitative RT-PCR and in situ hybridization on apc min/ þ and apc 1638N/ þ smad4 À/ þ tumours. Tumour cell lines stably transfected with LEF1 DN or APC2, or transiently transfected with short-interfering RNA (siRNA) against b-catenin showed a significant downregulation of ascl2. Colocalization of ascl2 with nuclear b-catenin was observed in 73 small intestinal adenocarcinomas (P ¼ 0.0008) and apc min/ þ tumours. Preliminary in vitro data suggest ascl2 may promote progression through the G2/M cell cycle checkpoint. In summary, ascl2 is a putative regulator of proliferation that is overexpressed in intestinal neoplasia.

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Section: Oligonucleotide Microarraysmentioning

confidence: 99%

Section: Oligonucleotide Microarraysmentioning

confidence: 99%

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Achaete-scute like 2 (ascl2) is a target of Wnt signalling and is upregulated in intestinal neoplasia

Jubb

Chalasani²,

Frantz³

et al. 2006

Oncogene

Self Cite

121

112

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“…The early growth of colorectal tumours requires angiogenesis (Goodlad et al, 2006;Korsisaari et al, 2007), the consequence of increased expression of pro-angiogenic factors (e.g., vascular endothelial growth factor-A (VEGF-A); Ferrara et al, 1991;Kim et al, 1993;Jubb et al, 2003;Korsisaari et al, 2007). The expression of VEGF in cancer is controlled by both oncogenic signalling (such as Wnt-signalling in colorectal cancer; Zhang et al, 2001) and hypoxia (Mizukami et al, 2004).…”

mentioning

confidence: 99%

“…The expression of VEGF in cancer is controlled by both oncogenic signalling (such as Wnt-signalling in colorectal cancer; Zhang et al, 2001) and hypoxia (Mizukami et al, 2004). Although there is redundancy among pro-angiogenic factors in advanced cancer (Relf et al, 1997;Hanrahan et al, 2003), many early cancers (Hanrahan et al, 2003;Jubb et al, 2003) and in vivo cancer models (Bergers et al, 1999;Hanrahan et al, 2003;Joyce et al, 2003;Goodlad et al, 2006;Korsisaari et al, 2007) are VEGF dependent. This observation has been exploited by the addition of an anti-VEGF monoclonal antibody (bevacizumab) to first-line chemotherapy in metastatic colorectal cancer, which prolonged the median overall survival (from 15.6 to 20.3 months, P ¼ 0.001; Hurwitz et al, 2004).…”

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confidence: 99%

Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer

Turley

et al. 2009

Br J Cancer

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Background: Delta-like ligand 4 (Dll4) is a Notch ligand that is upregulated by hypoxia and vascular endothelial growth factor-A (VEGF-A) and is reported to have a role in tumor angiogenesis. Evidence from xenograft studies suggests that inhibiting Dll4–Notch signalling may overcome resistance to anti-VEGF therapy. The aim of this study was to characterise the expression of Dll4 in colon cancer and to assess whether it is associated with markers of hypoxia and prognosis. Method: In all, 177 colon cancers were represented in tissue microarrays. Immunohistochemistry was performed using validated antibodies against Dll4, VEGF, hypoxia-inducible factor (HIF)-1 α , HIF-2 α , prolyl hydroxylase (PHD)1, PHD2, PHD3 and carbonic anhydrase 9 (CA9). Results: The expression of Dll4 was observed preferentially in the endothelium of 71% (125 out of 175) of colon cancers, but not in the endothelium adjacent to normal mucosa (none out of 107, P <0.0001). The expression of VEGF was significantly associated with HIF-2 α ( P <0.0001) and Dll4 ( P =0.010). Only HIF-2 α had a significant multivariate prognostic effect (hazard ratio 1.61, 95% confidence interval 1.01–2.57). Delta-like ligand 4 was also expressed by neoplastic cells, particularly neoplastic goblet cells. Conclusion: Endothelial expression of Dll4 is not a prognostic factor, but is significantly associated with VEGF. Assessing endothelial Dll4 expression may be critical in predicting response to anti-VEGF therapies.

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Plasmalemmal vesicle-associated protein (PLVAP) is expressed by tumour endothelium and is upregulated by vascular endothelial growth factor-A (VEGF)

Strickland¹,

Jubb²,

Hongo³

et al. 2005

J. Pathol.

104

105

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Vascular endothelial growth factor-A (VEGF) is an important regulator of vascular permeability. In preclinical studies, VEGF induces endothelial fenestrations in pre-existing and neo-vasculature, while inhibition of VEGF leads to a reduction in endothelial fenestrations. Recently, vascular regression in response to VEGF inhibition has been shown to correlate with the presence of endothelial fenestrations. Plasmalemmal vesicle-associated protein (PLVAP) is believed to be a component of diaphragmed endothelial fenestrations, but a direct relationship with VEGF signalling has not been established. The aim of this study was to characterize the expression pattern of PLVAP and investigate whether PLVAP is a transcriptional target of VEGF signal transduction. The expression pattern of PLVAP was characterized in normal and neoplastic human tissues by in situ hybridization and/or immunohistochemistry. The role of VEGF signal transduction in the regulation of PLVAP expression was investigated in vitro using receptor-selective engineered forms of VEGF, a neutralizing monoclonal antibody against VEGF, and inhibitors of downstream signalling pathways. PLVAP mRNA and protein were widely expressed in the endothelium of normal and neoplastic tissues. In cultured endothelial cells, VEGF signalling through receptor 2 stimulated expression of PLVAP total RNA and protein. This induction could be blocked with an anti-VEGF monoclonal antibody and by inhibitors of phosphatidylinositol 3-kinase (LY294002) or p38 mitogen-activated protein kinase (SB203580), but not by PD98059, a mitogen-activated protein/extracellular signal-regulated kinase 1 inhibitor. These data show that PLVAP is more widely expressed in the vasculature of normal tissues than previously thought and that it is expressed in the vasculature of most human tumours. We suggest that PLVAP is a downstream target of VEGF signalling. This work solidifies the association between VEGF and the appearance and maintenance of fenestrations by providing a potential mechanistic link.

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Quantitative analysis of colorectal tissue microarrays by immunofluorescence and in situ hybridization

Cited by 41 publications

References 51 publications

Achaete-scute like 2 (ascl2) is a target of Wnt signalling and is upregulated in intestinal neoplasia

Achaete-scute like 2 (ascl2) is a target of Wnt signalling and is upregulated in intestinal neoplasia

Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer

Plasmalemmal vesicle-associated protein (PLVAP) is expressed by tumour endothelium and is upregulated by vascular endothelial growth factor-A (VEGF)

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