Barbara A. Smith

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.

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.

Genomic Confirmation of Hybridisation and Recent Inbreeding in a Vector-Isolated Leishmania Population

Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the Çukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle.

Loss of cyclin-dependent kinase inhibitor genes and chromosome 9 karyotypic abnormalities in human bladder cancer cell lines

Loss of cell cycle control through the structural or functional aberration of checkpoint genes and their products is a potentially important process in carcinogenesis. In this study, a panel of well-characterised established human bladder cancer cell lines was screened by the polymerase chain reaction for homozygous loss of the cyclin-dependent kinase inhibitor genes p15, p16 and p27. The results demonstrate that, whereas there was no genetic loss of p27, homozygous deletion of both p15 and p16 genes occurred in seven of 13 (54%) independent bladder cell lines tested. Differential loss of either the p15 or p16 gene was not seen. The p15 and p16 genes are known to be juxtaposed on chromosome 9p21 at the locus of a putative tumour-suppressor gene involved in the initiation of bladder cancer. Cytogenetic analysis of the cell lines revealed karyotypes ranging from near diploid to near pentaploid with complex rearrangements of some chromosomes and a high prevalence of chromosome 9p rearrangements, although all cell lines contained at least one cytogenetically normal 9p21 region. These observations support a role for p15/p16 gene inactivation in bladder carcinogenesis and/or the promotion of cell growth in vitro and lend support to the hypothesis that homozygous deletion centred on 9p21 is a mechanism by which both p15 and p16 genes are co-inactivated.

Identification of Genes Up-Regulated in Urothelial Tumors: The 67-kd Laminin Receptor and Tumor-Associated Trypsin Inhibitor

Studies investigating changes in gene expression in urothelial carcinoma have generally compared tumors of different stages and grades but comparisons between low-grade, noninvasive tumors and normal urothelium are needed to identify genes involved in early tumor development. We isolated the urothelium from a low-grade tumor and corresponding normal mucosa by laser capture microdissection on frozen sections. The RNA extracted was amplified to generate suppressive subtractive cDNA libraries. Random sequencing of cDNA clones identified approximately 100 unique species. Of these 83% were known genes, 15% had homology to genes with an unknown function in humans, and 2% did not show homology to any published gene sequence. Two of the known genes, the 67-kd laminin receptor (67LR) and tumor-associated trypsin inhibitor (TATI), had previously been associated with metastatic progression in many tumor types, although 67LR has not been investigated in urothelial tumors. Immunolabeling of the original tissue with antibodies against these two genes confirmed overexpression, validating our strategy: 67LR was not expressed in the normal urothelium but was present in the tumor, whereas TATI expression was confined to umbrella cells in the normal urothelium, but extended to all cell layers in the tumor. We investigated both markers further in a separate series of tumors of different stages and grades. TATI was more consistently overexpressed than 67LR in all tumor grades and stages. Levels of secreted TATI were significantly higher in urine samples from patients with tumors compared to controls. Our strategy, combining laser capture microdissection and cDNA library construction, has identified genes that may be involved in the early phases of urothelial tumor development rather than with disease progression, highlighting the importance of comparing tumor with normal rather than just tumors of different stages and grades.

A role for the vesicle-associated tubulin binding protein ARL6 (BBS3) in flagellum extension in Trypanosoma brucei

The small GTPase Arl6 is implicated in the ciliopathic human genetic disorder Bardet–Biedl syndrome, acting at primary cilia in recruitment of the octomeric BBSome complex, which is required for specific trafficking events to and from the cilium in eukaryotes. Here we describe functional characterisation of Arl6 in the flagellated model eukaryote Trypanosoma brucei, which requires motility for viability. Unlike human Arl6 which has a ciliary localisation, TbARL6 is associated with electron-dense vesicles throughout the cell body following co-translational modification by N-myristoylation. Similar to the related protein ARL-3A in T. brucei, modulation of expression of ARL6 by RNA interference does not prevent motility but causes a significant reduction in flagellum length. Tubulin is identified as an ARL6 interacting partner, suggesting that ARL6 may act as an anchor between vesicles and cytoplasmic microtubules. We provide evidence that the interaction between ARL6 and the BBSome is conserved in unicellular eukaryotes. Overexpression of BBS1 leads to translocation of endogenous ARL6 to the site of exogenous BBS1 at the flagellar pocket. Furthermore, a combination of BBS1 overexpression and ARL6 RNAi has a synergistic inhibitory effect on cell growth. Our findings indicate that ARL6 in trypanosomes contributes to flagellum biogenesis, most likely through an interaction with the BBSome.

Novel urothelium specific gene expression identified by differential display reverse transcriptase-polymerase chain reaction

PURPOSE Understanding the molecular basis of differential gene expression among different tissues at various developmental stages and in neoplastic transformation is an important biological goal. The potential clinical applications of this improved understanding are more precise diagnosis of disease, prediction of prognosis, novel targeted therapies and prediction of response to therapy. MATERIALS AND METHODS Differential display reverse transcriptase-polymerase chain reaction was used to compare gene expression in bovine urothelium to that in autologous lung, esophagus, liver and spleen. Products that appeared to have urothelial specific expression were sequenced and assessed for homology with known sequences. Ribonuclease protection assays were used to further confirm the expression pattern. RESULTS A total of 32 discrete cDNAs were identified, including 3 products from genes known to be urothelium specific in their expression, 16 with significant homology to bovine, human or mouse expressed sequence tags and 5 with no sequence homology to any currently available sequence. Urothelium specific mRNA expression was confirmed for 3 genes by ribonuclease protection assays and one (Udd06) was further characterized as a urea transporter. CONCLUSIONS The use of differential display reverse transcriptase-polymerase chain reaction and other complementary techniques for parallel gene expression analysis will permit the complete characterization of the urothelial transcriptome and help identify potential molecular targets for rationally targeted therapy.

The Orthologue of Sjögren's Syndrome Nuclear Autoantigen 1 (SSNA1) in Trypanosoma brucei Is an Immunogenic Self-Assembling Molecule

Primary Sjögrens Syndrome (PSS) is a highly prevalent autoimmune disease, typically manifesting as lymphocytic infiltration of the exocrine glands leading to chronically impaired lacrimal and salivary secretion. Sjögrens Syndrome nuclear autoantigen 1 (SSNA1 or NA14) is a major specific target for autoantibodies in PSS but the precise function and clinical relevance of this protein are largely unknown. Orthologues of the gene are absent from many of the commonly used model organisms but are present in Chlamyodomonas reinhardtii (in which it has been termed DIP13) and most protozoa. We report the functional characterisation of the orthologue of SSNA1 in the kinetoplastid parasite, Trypanosoma brucei. Both TbDIP13 and human SSNA1 are small coiled-coil proteins which are predicted to be remote homologues of the actin-binding protein tropomyosin. We use comparative proteomic methods to identify potential interacting partners of TbDIP13. We also show evidence that TbDIP13 is able to self-assemble into fibril-like structures both in vitro and in vivo, a property which may contribute to its immunogenicity. Endogenous TbDIP13 partially co-localises with acetylated α-tubulin in the insect procyclic stage of the parasite. However, deletion of the DIP13 gene in cultured bloodstream and procyclic stages of T. brucei has little effect on parasite growth or morphology, indicating either a degree of functional redundancy or a function in an alternative stage of the parasite life cycle.