Phenotypic and Cytogenetic Characterization of Human Bladder Urothelia Expanded in Vitro

Cilento, Bartley G.; Freeman, Michael R.; Schneck, Francis X.; Retik, Alan B.; Atala, Anthony

doi:10.1016/s0022-5347(17)32676-9

Cited by 226 publications

(140 citation statements)

References 14 publications

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“…Because HB-EGF has been identified as an inflammatory cytokine and also because of its affinity for heparin-like glycosaminoglycans, we decided to examine the possibility that HB-EGF might play a role in normal bladder function. This hypothesis is supported by the recent identification of HB-EGF as an autocrine growth factor for human keratinocytes (36), as normal HUC proliferate avidly in media optimized for keratinocyte growth (24).…”

Section: Introductionmentioning

confidence: 75%

“…We have previously reported that normal HUC obtained from human bladder tissue can be expanded extensively under serum-free conditions in medium optimized for keratinocyte growth (24). EGF, which is used as a supplement for keratinocyte cultures, can be omitted from this medium with no detectable change in growth kinetics (24).…”

Section: Resultsmentioning

confidence: 99%

“…EGF, which is used as a supplement for keratinocyte cultures, can be omitted from this medium with no detectable change in growth kinetics (24). To determine if HUC cultured under these conditions synthesize HB-EGF, Northern blot analysis was performed.…”

Section: Resultsmentioning

confidence: 99%

“…Normal human urothelial cells were obtained from discarded human surgical material obtained from donors between the ages of 2 and 10 yr as described previously (24). Cells were cultured and passaged in K-SFM (GIBCO BRL, Grand Island, NY) serum-free medium supplemented with 50 mg/ml bovine pituitary extract (BPE).…”

Section: Methodsmentioning

confidence: 99%

“…Increased HER1 expression has also been linked spatially to local increases in cell proliferation (21), suggesting a role for one or more HER1 ligands in aberrant urothelial cell growth. However, several reports have shown that EGF does not promote the growth of normal human urothelial cells (HUC) in serum-free medium, despite the presence of high-affinity EGF binding sites in these cells (22)(23)(24). In contrast, human TCC cell lines have been shown to proliferate in response to EGF (22,23).…”

Section: Introductionmentioning

confidence: 99%

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Heparin-binding EGF-like growth factor is an autocrine growth factor for human urothelial cells and is synthesized by epithelial and smooth muscle cells in the human bladder.

Freeman¹,

et al. 1997

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The epidermal growth factor receptor (HER1) has been implicated in regenerative growth and proliferative diseases of the human bladder epithelium (urothelium), however a cognate HER1 ligand that can act as a growth factor for normal human urothelial cells (HUC) has not been identified. Here we show that heparin-binding EGF-like growth factor (HB-EGF), an activating HER1 ligand, is an autocrine regulator of HUC growth. This conclusion is based on demonstration of HB-EGF synthesis and secretion by primary culture HUC, identification of HER1 as an activatable HB-EGF receptor on HUC surfaces, stimulation of HUC clonal growth by HB-EGF, inhibition of HB-EGF-stimulated growth by heparin and of log-phase growth by CRM 197, a specific inhibitor of HB-EGF/HER1 interaction, and identification of human urothelium as a site of HB-EGF precursor (proHB-EGF) synthesis in vivo. ProHB-EGF expression was also detected in the vascular and detrusor smooth muscle of the human bladder. These data suggest a physiologic role for HB-EGF in the regulation of urothelial proliferation and regeneration subsequent to mucosal injury. Expression of proHB-EGF is also a feature of differentiated vascular and detrusor smooth muscle in the bladder. Because proHB-EGF is known to be the high affinity diphtheria toxin (DT) receptor in human cells, synthesis of the HB-EGF precursor by human urothelium also suggests the possibility of using the DT-binding sites of proHB-EGF as an in vivo target for the intraluminal treatment of urothelial diseases. ( J. Clin. Invest. 1997. 99:1028-1036.)

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Section: Introductionmentioning

confidence: 75%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Heparin-binding EGF-like growth factor is an autocrine growth factor for human urothelial cells and is synthesized by epithelial and smooth muscle cells in the human bladder.

Freeman¹,

et al. 1997

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Expression and functions of EGF FGF and TGFβ-growth-factor family members and their receptors in invasive human transitional-cell-carcinoma cells

et al. 1997

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Growth factors such as transforming growth factor beta(TGFb), epidermal growth-factor(EGF) and fibroblast-growth-factorlike(FGF) proteins have been reported to be implicated in wound healing, tumorigenesis and embryogenesis. These proteins are capable of modulating, e.g., cellular proliferation, differentiation, migration or protein synthesis. EGF-like proteins, including EGF, TGFa, amphiregulin and heregulins (HRG), are expressed in various normal epithelial and carcinoma cells (Plowman et al., 1990;Johnson et al., 1992;Danilenko et al., 1995;Salomon et al., 1995). In several carcinomas, such as breast and renal carcinomas, over-expression of TGFa and/or amphiregulin has been observed (Mydlo et al., 1989;LeJeune et al., 1993;Salomon et al., 1995). Normal human bladder urothelium is reported to express EGF, TGFa and amphiregulin, while high levels of EGF have been found in urine of normal individuals (Kimball et al., 1984;Lau et al., 1988;Cilento et al., 1994). However, the urine of patients with transitional-cell carcinoma (TCC) contains enhanced levels of TGFa (Kimball et al., 1984). Expression of the EGF receptor (EGFR) which binds EGF, TGFa and amphiregulin, was demonstrated to be augmented in human TCCs, notably in the more aggressive TCCs (Messing et al., 1987;Neal et al., 1990). Functional effects of EGF-like proteins include stimulation of proliferation and migration of different normal epithelial and carcinoma cells, including murine urothelial cells (De Boer et al., 1993Danilenko et al., 1995). Both expression and functional data suggest a direct function for EGF-like molecules in TCC development or progression. While HRG were shown to enhance proliferation, migration or differentiation of normal skin and breast epithelial cells Marikovsky et al., 1995;Yang et al., 1995), less is known about the functions of HRG in tumour growth. The expression of their receptors, c-erbB3 (HER3) and c-erbB4 (HER4), was observed to be enhanced in several tumours, including breast and bladder carcinomas (Lemoine et al., 1992;Plowman et al., 1993;Rajkumar et al., 1996), suggesting an implication of HRG in these tumours. While c-erbB3 was also reported to be present in the normal urogenital tract, little is known about functions of HRG or their receptors in human urothelium or TCCs.FGF have also been suggested to be involved in human TCC growth. Chopin et al. (1993) demonstrated enhanced FGF-1, but not FGF-2, expression in TCCs. Furthermore, FGF-1 was shown to induce in vitro proliferation, motility and invasion of murine bladder-carcinoma cells (Vallés et al., 1990;Tucker et al., 1991;De Boer et al., 1993). These studies suggest a direct role for FGF-1 in TCC-cell invasion. Finally, mis-sense mutations in TGFb receptors(TGFbR) types I and II have been shown in colon-, gastric-and prostate-carcinoma cells (Myeroff et al., 1995;Lee et al., 1996), resulting in TGFb-non-responsive cells . Although functional TGFb receptors(TGFbR) types I and II are expressed in normal human urothelium (De Boer et al., 1996), little is known a...

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MHC-dependent cytolysis of autologous tumor cells by lymphocytes infiltrating urothelial carcinomas

Housseau¹,

Zeliszewski²,

Roy³

et al. 1997

Int. J. Cancer

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Tumor-infiltrating lymphocytes (TIL) were grown from 23 urothelial carcinomas. Phenotyping analysis showed that the TIL cultures were mainly CD3+. Although CD4+ and CD8+ T-cell sub-sets were grown in culture, CD4+ T-cell sub-sets predominated over CD8+ T cells. Immunohistochemical studies performed on 5 tumor specimens confirmed this observation, and indicated that CD4+ T cells surrounded the tumor islets, whereas CD8+ T lymphocytes were localized among the tumor cells. Five short-term carcinoma cell lines established from these urothelial tumors were used as target cells in cytolysis assays in order to investigate the functional anti-tumor activity of autologous TIL. TIL from 4/5 tumors were lytic and 3 TIL lines displayed MHC-class-I-dependent cytotoxicity directed against autologous tumor cells. CD4+ T-cell-depletion experiments performed on TIL line 07 confirmed that CD8+ MHC-class-I-dependent CTL were the predominant effectors. Finally, experiments performed on 6 allogeneic urothelial-cancer cell lines matched for HLA-class-I molecules showed that TIL07 exhibited selective lytic activity toward tumor 07. These data indicate that CD8+ MHC-class-I-dependent CTL present in urothelial carcinomas are functional and may participate in the anti-tumor immune response.

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Phenotypic and Cytogenetic Characterization of Human Bladder Urothelia Expanded in Vitro

Cited by 226 publications

References 14 publications

Heparin-binding EGF-like growth factor is an autocrine growth factor for human urothelial cells and is synthesized by epithelial and smooth muscle cells in the human bladder.

Heparin-binding EGF-like growth factor is an autocrine growth factor for human urothelial cells and is synthesized by epithelial and smooth muscle cells in the human bladder.

Expression and functions of EGF FGF and TGFβ-growth-factor family members and their receptors in invasive human transitional-cell-carcinoma cells

MHC-dependent cytolysis of autologous tumor cells by lymphocytes infiltrating urothelial carcinomas

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