Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-01-16
2003-02-04
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S350000, C530S387100
Reexamination Certificate
active
06514708
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a process for detecting the malignant transformation of cells and its application for diagnosing and monitoring cancers and for developing cancer prognosis.
BACKGROUND OF THE INVENTION
Chorionic gonadotropic hormone (hCG) is a glycoprotein which is formed from two subunits which are termed alpha (hCG&agr;) and beta (hCG&bgr;) and which are linked non-covalently (1). During pregnancy, the trophoblastic cells of the placenta produce the dimeric hCG and the free hCG&agr; or hCG&bgr; subunits which are found in substantial quantity in the serum. The development of sensitive and. specific techniques for assaying hCG and, independently, for assaying the free hCG&agr; subunit and the free hCG&bgr; subunit, made it possible to show that serum levels of hCG of up to 1000 pg/ml and serum levels of the free hCG&agr; subunit of up to 3000 pg/ml were present in non-pregnant healthy subjects (2,3). It was also observed that serum levels of hCG or of the free hCG&agr; subunit greater than these normal values are principally to be found in patients having a testicular or ovarian tumor of trophoblastic origin.
By contrast, only low serum levels of the free hCG&bgr; subunit, i.e. which are less than 100 pg/ml, can be detected in non-pregnant healthy subjects. Serum levels of free hCG&bgr; which are greater than these normal values are found in a large number of cancer patients who are carrying a tumor of gonadal or non-gonadal origin (2). In particular, 47% of patients with cancer of the bladder, 32% of patients with cancer of the pancreas and 30% of patients with cancer of the uterine cervix have a serum level of free hCG&bgr; which is greater than 100 pg/ml (2). In patients with cancer of the bladder, the presence of an elevated level (greater than 100 pg/ml) of free hCG&bgr; is reported to be correlated with unfavorable development of the disease (4,5).
While the hCG&agr; subunit is encoded by one single gene, which is located on chromosome 6q21.1-q23, the hCG&bgr; subunit has been reported to be encoded by a family of genes which are located on chromosome 19q13.3. Following a large number of studies, it was shown that there were 7 CG&bgr; genes, termed CG&bgr;7 or &bgr;7, CG&bgr;6 or &bgr;6, CG&bgr;8 or &bgr;8, CG&bgr;5 or &bgr;5, CG&bgr;1 or &bgr;1, CG&bgr;2 or &bgr;2 and CG&bgr;33 or &bgr;3 (6). The &bgr;6 and &bgr;7 genes are allelic (7).
Only the &bgr;7, &bgr;6, &bgr;8, &bgr;5 and &bgr;3 genes are able to encode the hCG&bgr; subunit, which is made up of 145 amino acids. The &bgr;1 and &bgr;2 genes are characterized by the presence:
of an insertion of approximately 770 nucleotides in the 5′ part of the CG&bgr; genes,
of a point mutation of the 5′ splicing site of the first intron of the CG&bgr; genes.
It has been shown that the p1 and &bgr;2 genes are capable of being transcribed in some tissues and would be able to encode a protein of 132 amino acids having a different sequence from that of hCG&bgr; (7).
Sequencing all or part of the p7, &bgr;6, &bgr;5 and &bgr;3 genes, and the restriction maps, showed that the nucleotides corresponding to codons 2, 4 and 117 with respect to the mature CG&bgr; protein were different depending on the genes in question (8, 9, 10).
Analysis of the nucleotide sequences for equivalent positions was carried out in the case of the &bgr;2 gene and it was shown that the gene exhibited the same characteristics as the &bgr;6 gene. On the other hand, only position 117 of the &bgr;1 gene was deduced by restriction map analysis and was regarded as being “of the Asp type”.
In addition, expression of the different CG&bgr; genes in placental tissue has been analyzed in a semiquantitative manner. The results demonstrate that the &bgr;5 transcripts are in much higher abundance than the &bgr;3 and &bgr;8 transcripts. The &bgr;7, &bgr;1 and &bgr;2 transcripts are very much in the minority (7). Finally, several studies have been carried out with the aim of looking for CG&bgr; transcripts in various normal or neoplastic nontrophoblastic tissues or on various cell lines derived from cancerous tissues of trophoblastic or nontrophoblastic origin (11-17). The techniques employed in these different studies did not distinguish between the &bgr;7, &bgr;8, &bgr;5 and &bgr;3 transcripts. These studies demonstrated that &bgr;7, &bgr;8, &bgr;5 or &bgr;3 transcripts are present, in particular, in normal testes (11), neoplastic testes (12), neoplastic bladder (13), normal placenta, choriocarcinomic placental cell lines (14-16) and in various cells lines derived from neoplastic nontrophoblastic cells (15-17).
Recently, a study was carried out on normal and neoplastic tissues of vesical origin for the purpose of quantifying the &bgr;7, &bgr;8, &bgr;5 and &bgr;3 transcripts. This study was based on the profile observed after amplifying exons 1 and 2 and enzymatically cleaving in nucleotide positions corresponding to the 5′ transcribed, untranslated part of exon 1 (18). This study demonstrated that normal vesical tissue only expresses the &bgr;7 gene and that malignant transformation of vesical tissue is accompanied by acquisition of the ability to express the &bgr;8 and/or &bgr;5 and/or &bgr;3 genes in 45 to 95% of cases (18).
SUMMARY OF THE INVENTION
The present invention is based on demonstrating the importance of the &bgr;3, &bgr;5, &bgr;8 and &bgr;9 genes in the progression to malignancy, in particular as compared with the expression, which exists in normal tissues, of the products of the &bgr;6 and &bgr;7 genes.
The present invention relates to a process for detecting the malignant transformation of human cells, characterized in that overexpression of the products of the &bgr;3, &bgr;5, &bgr;8 and/or &bgr;9 genes, encoding the hCG&bgr; subunit, as compared with their expression in nonmalignant cells, is demonstrated in the said cells.
REFERENCES:
patent: 94/20859 (1994-09-01), None
Lockhart et al., Nature, vol. 405, pp 827-836; (2000).*
Marcillac et al., Cancer Research; vol. 52, pp 3901-3907; 1992.*
V. Lazar et al., “Cancer Research”,Expression of Human Chorionic Gonadotrophin Beta Subunit Genes in Superficial and Invasive Bladder Carcinomas, vol. 55, No. 17, pp. 3735-3738, (Sep. 1, 1995).
G.H. Cotton, “The Biochemical Journal”,Detection of Single Base Changes in Nucleic Acids, vol. 263, pp. 1-10, (Oct. 1989).
F. Housseau et al., “International Journal of Cancer”,Reaction of Peripheral Blood Lymphocytes to the Human Chorionic Gonadotrophin Beta Sub-Unit in Patients with Productive Tumors, vol. 63, pp. 633-638, (Nov. 27, 1995).
J. Bidart et al., Trends in Endocrinol. Metabolism,Human Chorionic Gonadotrophin: Molecular Forms, Detection, and Clinical Implications, vol. 4, No. 9, pp. 285-291, (Nov. 1993).
D. Bellet et al., “Cancer Research”,Malignant Transformation of Nontrophoblastic Cells is Associated with the Expression of Chorionic Gonadotropin Beta Genes Normally Transcribed in Trophoblastic Cells, vol. 57, (Jan. 1997).
Forough et al (1993) J. Biol. Chem. 268: 2960-2968.
Andreadis et al (1987) Ann. Rev. Cell Biol. 3: 207-242.
Bellet Dominique
Bidart Jean-Michel
Lazar Vladimir
Vidaud Michel
Foley & Lardner
Institut Gustave Roussy
Jones W. Gary
Souaya Jehanne
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