Jennifer Southgate

Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction

Only small numbers of cells from solid tumours are needed for haematogenous metastasis. Detection is difficult because existing techniques are not sensitive enough. We have used reverse transcriptase to make complementary DNA from peripheral blood messenger RNA, and the polymerase chain reaction (PCR) to amplify cDNA specific for a gene actively transcribed only in the tumour tissue type. We prepared cDNA from peripheral blood of seven patients with malignant melanoma, four patients with other metastatic cancers, and four healthy subjects, as well as from several melanoma-derived cell lines. PCR was used to amplify the gene for tyrosinase, a tissue-specific gene in melanocytes. Since normal melanocytes are not thought to circulate in peripheral blood, detection of tyrosinase transcription in peripheral blood should indicate the presence of circulating cancer cells. The method was highly sensitive and could detect a single melanoma cell from a cell line in 2 ml normal blood. Blood samples from four of the seven patients with malignant melanoma gave positive results, whereas all eight control subjects gave negative results. This method does not depend on the characterisation of cancer-specific genetic abnormalities and can be applied to any cancer for which tissue-specific genes can be identified, including epithelial cancers. It could prove useful in the diagnosis of primary or metastatic cancers, in assessing prognosis, and in detecting residual disease after treatment.

Multistage carcinogenesis induced by ras and myc oncogenes in a reconstituted organ

ras and myc oncogenes were able to induce distinct phenotypic alterations, resembling different types of premalignant lesions, when introduced into approximately 0.1% of the cells used to reconstitute the mouse prostate gland. While ras induced dysplasia in combination with angiogenesis, myc induced a hyperplasia of the otherwise normally developed organ. ras and myc together induced primarily carcinomas. However, tumor progression was also associated with additional genetic alterations involving gene amplification. Our data indicate that specific types of benign premalignant lesions may reflect the activation of different single oncogenes, and that the consecutive activation of multiple oncogenes could be a causal event in the step-like progression of tumorigenesis.

Regional copy number–independent deregulation of transcription in cancer

Genetic and epigenetic alterations have been identified that lead to transcriptional deregulation in cancers. Genetic mechanisms may affect single genes or regions containing several neighboring genes, as has been shown for DNA copy number changes. It was recently reported that epigenetic suppression of gene expression can also extend to a whole region; this is known as long-range epigenetic silencing. Various techniques are available for identifying regional genetic alterations, but no large-scale analysis has yet been carried out to obtain an overview of regional epigenetic alterations. We carried out an exhaustive search for regions susceptible to such mechanisms using a combination of transcriptome correlation map analysis and array CGH data for a series of bladder carcinomas. We validated one candidate region experimentally, demonstrating histone methylation leading to the loss of expression of neighboring genes without DNA methylation.

The relationship between the mechanical properties and cell behaviour on PLGA and PCL scaffolds for bladder tissue engineering

Previous work on 2D synthetic films showed growth of human bladder stromal cells was enhanced on materials with lower moduli that mimic the elastic properties of native tissue. This study developed 3D synthetic foam scaffolds for soft tissue engineering by emulsion freeze-drying. Foams of poly(lactide-co-glycolide) (PLGA) and poly(epsilon-caprolactone) (PCL) were extensively characterised using scanning electron microscopy, mercury porosimetry, dynamic mechanical analysis, degradation analysis, size exclusion chromatography and differential scanning calorimetry. Foams of 85-88% porosity and 35 microm pore diameter were selected for further study; the storage modulus of PCL foams was around half that of PLGA (2 MPa vs 4 MPa) and closer to the reported value for native bladder tissue. Urinary tract stromal cells showed a 4.4 and 2.4-fold higher attachment and rate of growth, respectively, on PCL scaffolds, as assessed by a modified 3-[4,5-dimethyl(thiazol-2yl)-3,5-diphery] tetrazolium bromide assay. A greater contractile force was exerted by cells seeded in PLGA than on PCL scaffolds, raising the possibility that the reduced rate of proliferation of cells on PLGA scaffolds may reflect differentiation into a contractile phenotype. This study has generated PCL foam scaffolds with properties that may be pertinent to the tissue engineering of the bladder and other soft tissues.

Reconstitution of Human Urothelium From Monolayer Cultures

PURPOSE We established a 3-dimensional organ culture model of urinary tract tissue in which to study the effects of seeding cultured urothelial cells onto de-epithelialized urothelial stroma. MATERIALS AND METHODS Normal human urinary tract tissues were placed in organ culture or used to establish urothelial cell cultures. At passage 2 cell cultures were harvested and used to reconstitute autologous organ cultures by seeding onto de-epithelialized stroma. Organ cultures were harvested at intervals and analyzed by immunohistology with a panel of antibodies against differentiation associated antigens, cytokeratins, cell adhesion molecules, extracellular matrix components and proliferation associated antigens. RESULTS Human urothelial tissues were maintained in organ culture for at least 18 weeks and they retained a transitional epithelial morphology with expression of normal in situ antigenic characteristics. Within 2 weeks of reconstitution recombined organ cultures formed a stratified, polarized, transitional-like neo-epithelium that expressed many of the phenotypic and differentiated characteristics of normal tissue. Basement membrane formed at sites of direct contact between urothelial cells and stroma. After an initial stabilization period the proliferation rate of the urothelium of intact and reconstituted organ cultures decreased to the low turnover rate characteristic of normal urothelium in situ, indicating that the cells were responsive to normal growth regulatory controls. CONCLUSIONS Normal human urothelial cells, which express a proliferative nondifferentiated phenotype in monolayer culture, retain the capacity to differentiate and reform a slow turnover, stratified transitional epithelium.

Uroplakin gene expression in normal human tissues and locally advanced bladder cancer

The uroplakins are widely regarded as urothelium‐specific markers of terminal urothelial cytodifferentiation. This study investigated the expression of the four uroplakin genes, UPIa, UPIb, UPII and UPIII, in a wide range of normal human tissues to determine tissue specificity and in advanced transitional cell carcinoma (TCC) to examine gene expression in primary and metastatic disease. In the urinary tract, all four uroplakins were expressed by urothelium and UPIII was also expressed by prostatic glandular epithelium. UPIa and UPII appeared to be urothelium‐specific, but UPIb was detected in several non‐urothelial tissues, including the respiratory tract, where it was associated with squamous metaplasia of tracheal and bronchial epithelia. The ten cases of primary TCC and corresponding lymph node metastases demonstrated that each uroplakin gene could be expressed at the mRNA level. No single uroplakin gene was expressed in all primary tumours or metastases, but 80% of the primary tumours and 70% of the lymph node metastases expressed at least one uroplakin gene. UPIII mRNA was often expressed in the absence of UPIII protein. These results confirm that in human tissues the expression of UPIa and UPII genes is highly specific to urothelium and suggest that the tight differentiation‐restricted expression of uroplakin genes in normal urothelium is lost following malignant transformation. Copyright

Uroplakin Gene Expression by Normal and Neoplastic Human Urothelium

cDNA sequences for human uroplakins UPIa, UPIb, UPII, and UPIII were cloned and used to investigate uroplakin transcription by normal and neoplastic urothelial cells. Normal urothelium expressed mRNA for all four uroplakins, although UPIII could be detected only by ribonuclease protection assay. By in situ hybridization, UPIa and UPII were confined to superficial cells and UPIb was also expressed by intermediate cells. Cultured normal human urothelial cells showed a proliferative basal/intermediate cell phenotype and constitutive expression of UPIb only. Uroplakin expression by transitional cell carcinoma cell lines was related to their differentiated phenotype in vitro. RT4 cells expressed all uroplakins, VM-CUB-3 expressed three uroplakins, RT112 and HT1376 cells expressed only UPIb in high abundance, and COLO232, KK47, and EJ cells had no detectable expression. These results correlated with patterns of uroplakin expression in tumors. UPIa and UPII were detected superficially only in well differentiated transitional cell carcinoma papillae. UPIb was positive in seven of nine and overexpressed in five of nine noninvasive transitional cell carcinomas and was also present in four of eight invasive transitional cell carcinomas. Lymph node metastases retained the same pattern of UPIb expression as the primary tumor. Unlike the three differentiation-regulated uroplakins, UPIb may have an alternative role in urothelial cell/tissue processes.

Role of PPARgamma and EGFR signalling in the urothelial terminal differentiation programme

Recently, considerable interest has focused on the ability of activated peroxisome proliferator-activated receptor γ (PPARγ) to promote cytodifferentiation in adipocytes and some carcinoma cells; however, the role of PPARγ in normal epithelial cytodifferentiation is unknown. Using uroplakin (UP) gene expression as a specific correlate of terminal urothelial cytodifferentiation, we investigated the differentiation-inducing effects of PPARγ activation in normal human urothelial (NHU) cells grown as finite cell lines in monoculture. Two high-affinity activators of PPARγ, troglitazone (TZ) and rosiglitazone (RZ) induced the expression of mRNA for UPII and UPIb and, to a lesser extent, UPIa. The specificity of the effect was shown by pretreating cells with a PPARγ antagonist, GW9662, which attenuated the TZ-induced response in a dose-specific manner. The PPARγ-mediated effect on UP gene expression was maximal when there was concurrent inhibition of autocrine-activated epidermal growth factor receptor (EGFR) signalling through either the phosphatidylinositol 3-kinase or extracellular signal-regulated kinase (ERK) pathways. The use of a specific EGFR tyrosine kinase inhibitor, PD153035, correlated with PPARγ dephosphorylation and translocation to the nucleus, indicating a mechanism for regulating the balance between proliferation and differentiation. This is the first identification of specific factors involved in regulating differentiation-associated gene changes in urothelium and the first unambiguous evidence of a role for PPARγ signalling in the terminal differentiation programme of a normal epithelium.

Agent-based computational modeling of wounded epithelial cell monolayers

Computational modeling of biological systems, or in silico biology, is an emerging tool for understanding structure and order in biological tissues. Computational models of the behavior of epithelial cells in monolayer cell culture have been developed and used to predict the healing characteristics of scratch wounds made to urothelial cell cultures maintained in low- and physiological [Ca/sup 2+/] environments. Both computational models and in vitro experiments demonstrated that in low exogenous [Ca/sup 2+/], the closure of 500-/spl mu/m scratch wounds was achieved primarily by cell migration into the denuded area. The wound healing rate in low (0.09 mM) [Ca/sup 2+/] was approximately twice as rapid as in physiological (2 mM) [Ca/sup 2+/]. Computational modeling predicted that in cell cultures that are actively proliferating, no increase in the fraction of cells in the S-phase would be expected, and this conclusion was supported experimentally in vitro by bromodeoxyuridine incorporation assay. We have demonstrated that a simple rule-based model of cell behavior, incorporating rules relating to contact inhibition of proliferation and migration, is sufficient to qualitatively predict the calcium-dependent pattern of wound closure observed in vitro. Differences between the in vitro and in silico models suggest a role for wound-induced signaling events in urothelial cell cultures.

Expression of cytokeratin 20 redefines urothelial papillomas of the bladder

BACKGROUND Most non-invasive urothelial tumours of the bladder are diagnosed as papillary carcinomas in accordance with the WHO classification and because the identification of papillomas is difficult by routine histology; some patients are therefore misdiagnosed. This practice is associated with psychological morbidity for the patient and may also skew cancer statistics. Cytokeratin 20 (CK20) is a sensitive marker of urothelial differentiation. We investigated whether this marker could be used in the identification of urothelial papillomas and used the rate of recurrence as an indicator to assess the biological behaviour of such tumours. METHODS In a prospective study, immunocytochemistry for CK20 was done on tumours of all patients who presented for the first time with non-invasive papillary bladder tumours. We classified the expression pattern of CK20 as normal or abnormal at the time of initial diagnosis. We recorded time to first biopsy-proven recurrence or length of follow-up when no recurrence was observed. FINDINGS Of 58 consecutive patients, ten had tumours with a normal pattern of CK20 expression. No patients developed further tumours during the follow-up (median 18 [range 13-28] months). By contrast, 30 (73%) of the 41 evaluable patients with tumours that showed abnormal CK20 expression developed further tumours; the median time to a second tumour was 6 (2-24) months. The only factor that had a significant effect on the outcome of patients in terms of recurrence was expression of CK20 (p<0.0001). INTERPRETATION Normal urothelial differentiation, as evidenced by a normal pattern of CK20 expression, is retained in a proportion of non-invasive papillary urothelial tumours and thus justifies use of the term urothelial papilloma. A large-scale study is needed to investigate the outcome of patients with such tumours.