Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-11-13
2001-08-21
Horlick, Kenneth R. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023500, C435S006120, C435S007100, C435S091100, C435S091200
Reexamination Certificate
active
06277968
ABSTRACT:
BACKGROUND OF THE INVENTION
Histological differentiation markers are useful in the diagnosis of carcinoma metastases where the location of the primary tumor is uncertain or unknown. Unfortunately, markers specific for a single epithelium or organ are currently available for a only few types of carcinoma, e.g., prostate-specific antigen for prostate carcinomas and thyroglobulin for thyroid carcinomas.
Less specific markers of transitional cell carcinomas have been identified and associated with malignant transformation, tumor progression and the prognosis. Many of these markers are epithelial membrane antigen (EMA) or oncogene/tumor suppressor gene products. For example, Summerhayes et al. (1985
. JNCI
75:1025-1038) have described a series of monoclonal antibodies (group III), directed against the urothelium which produce luminal-membrane staining of normal superficial (umbrella) cells of the urothelium. Other markers are expressed in superficial bladder tumors but disappear in invasive and metastatic transitional cell carcinomas. All of these markers are antibodies most of which stain non-urinary epithelia and carcinomas too. Certain antigens such as involucrin, E48 antigen and SCC antigen are markers shared by both transitional and stratified squamous epithelia (of skin, esophagus, cervix, etc. ) and their carcinomas. However, no differentiation or lineage marker specific for transitional cell carcinomas and their metastases has been identified to date.
Normal urothelium contains tissue-specific differentiation products that have been well characterized morphologically and biochemically. It has been found that large numbers of urothelial plaques are present in the superficial plasma membrane of urothelial superficial or umbrella cells. These plaques are characterized by a highly unusual membrane structure, i.e., the asymmetric unit membrane (AUM), whose luminal leaflet is twice as thick as its cytoplasmic leaflet. The thickening of the luminal leaflet is due to the presence of particles exhibiting a semi-crystalline organization. The molecular constituents principally comprise four transmembrane proteins: uroplakin (UP) Ia (27 kDa); UP Ib (28 kDa); UP II (15 kDa) and UP III (47 kDa). These UPs, particularly UP Ia, Ib, and II, are characterized by their markedly asymmetric mass distribution, with the extracellular domain being considerably larger than the intracellular one. This accounts for the clearly visible ultrastructural thickening of the luminal leaflet of the unit membrane. UP III is believed to play a role in the formation of the urothelial glycocalyx and may interact, via its cytoplasmic portion, with the cytoskeleton.
The identification and characterization of the uroplakins and their role as molecular markers has been described by Yu et al. 1990
. J. Cell Biol
. 111:1207-1216; Wu et al. 1990
. J. Biol. Chem
. 265:19170-19179; Wu et al. 1993
. J. Cell Sci
. 106:31-43; Wu et al. 1994
. J. Biol. Chem
. 269:13716-13724; Yu et al. 1994
. J. Cell Biol
. 125:171-182; and Lin et al. 1994
. J. Biol. Chem
. 269:1775-1784. Further, Ryan et al. (1993
. Mammalian Genome
4:656-661) describe mapping the chromosomal locations of mouse and bovine UP Ia and UP Ib and suggest likely locations for human genes. The locations of human UP II, UP IIIa and UP IIIb genes are also predicted. Ryan et al. suggest that uroplakin genes could be involved in chronic urinary tract diseases and also suggests diagnostic uses (e.g., interstitial cystitis). Mono-specific antibodies to UP Ia, UP Ib, UP II and UP III were used to show that uroplakin expression is confined to the urinary bladder and to determine the location of UP Ia, UP Ib, UP II and UP III within the asymmetric unit membrane. However, identification, sequencing, or chromosomal location of the human gene for any of the uroplakins has not yet been disclosed.
Yu et al. (1992
. Epith. Cell Biol
. 1:4-b 12
) describe the use of specific antibodies to AE31 and AE32 for analysis of the differentiation state of bovine urothelial cells. The diagnostic use of antibodies to uroplakins to positively identify metastatic bladder carcinomas has also been described by Moll, et al. 1993
. Verh. Deutsc. Ges. Path
. 77 and Moll, R. et al. 1995
. Am. J. Pathol
. 147:1383-1397.
The human gene for uroplakin II has now been isolated and sequenced. Using the knowledge of the human uroplakin gene sequence a highly specific uroplakin-based reverse transcriptase (RT)-polymerase chain reaction (PCR) assay has now been developed which is useful in the detection of circulating bladder cancer cells in metastatic bladder cancer patients.
SUMMARY OF THE INVENTION
UP Ia, Ib, II and III are the first molecular markers that have been found to be specific for urothelial differentiation. The human gene sequence of UP II has now been determined. Knowledge of this sequence has been used to develop molecular probes, primer sequences, that are used in a RT-PCR assay to detect the presence of bladder cancer cells in blood and tissues. Accordingly, the assay of the present invention is useful in identifying and diagnosing metastatic bladder cancer cells. Further, the RT-PCR assay can be used to identify mutations in the uroplakin genes.
DETAILED DESCRIPTION OF THE INVENTION
UPs Ia, Ib, II and III are specialized membrane proteins of the urothelial plaque constituting the AUM and represent the first specific molecular markers of urothelial differentiation. UPs are widely conserved with respect to their structural organization and amino acid sequence among all mammals. UP II and III have been found to be immunohistochemically detectable in routinely prepared paraffin sections of human urothelium. Extensive UP III screening of a variety of normal tissues revealed that the urothelium-specificity of this glycoprotein, which until now has been documented only in bovine tissues, is also valid for human tissues. Unlike UP Ia and UP Ib which have 4 putative transmembrane domains (Yu, J. et al. 1994
. J. Cell Biol
. 125:171-182), UP II and UP III have only one transmembrane domain (Lin, J. H. et al. 1994
. J. Biol. Chem.
269:1775-1784; Wu, X. R. and T. T. Sun. 1993
. J. Cell Sci.
106:31-43). Recent data indicate that UP II and UP III are preferentially crosslinked to UP Ia and UP Ib, respectively, suggesting the existence of two types of 16 nm AUM particles consisting of UP II/UP Ia and UP III/UP Ib (Wu, X. R. et al. 15 1995
. J. Biol. Chem
. 270:29752-29759).
Mouse and bovine UP II genes have been sequenced. The cDNA-derived amino acid sequences of the mouse and bovine UP II are 83% identical thus indicating a high degree of structural and possibly functional conservation (Wu, X. R. et al. 1995
. J. Biol. Chem
. 270:29752-29759).
The human UP II gene has also now been sequenced. In these experiments, a human genomic library in lambda Fix-II phage (Stratagene, La Jolla, Calif.) was screened with a
32
P-labeled bovine uroplakin II cDNA (Lin, J. H. et al. 1994
. J. Biol. Chem.
269:1775-1784). Two positive clones were identified. The clone having a longer 5′-flanking sequence was then further characterized. Two Sac I fragments (3.3 kb and 5 kb), that together contained the entire coding region of human uroplakin II gene, were subcloned and sequenced. This sequence is shown as SEQ ID NO: 1. The gene contains 5 exons spanning approximately 2 kb, similar to the mouse and bovine genes. Its deduced amino acid sequence, shown as SEQ ID NO: 2, is 79% identical to those of the mouse and bovine analogues, consisting of a hydrophobic N-terminal signal peptide (approximately 25 amino acid residues) and a prosequence (approximately 59 amino acid residues) harboring 3 potential N-glycosylation sites, and ending with a RGRR cleavage site for furin, which may be involved in UP II processing and maturation, and a mature protein (100 residues) with a C-terminal hydrophobic potential transmembrane domain (approximately 25 residues).
The chromosomal location of human UP II gene has also now been determined. In these experiments, a 21 kb fragment of the human UP II gene containing all five exons was
Sun Tung-Tien
Wu Xue-Ru
Browdy and Neimark
Horlick Kenneth R.
New York University
Siew Jeffrey
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