Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-01
2001-08-21
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06277581
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates, in a general sense, to assessing the susceptibility of mammals to carcinogenesis.
An individual's susceptibility to cancer is governed by the individual's genome and carcinogenic stimuli which the individual encounters in the environment. Although the carcinogenic potentials of many compounds and other stimuli (e.g. ionizing radiation) have been determined, assessing the importance of limiting exposure to such compounds is complicated by the fact that not all individuals are equally susceptible to the carcinogenic effects of the compounds. Thus, the genetic component of carcinogenic susceptibility limits accurate prediction of cancer rates among individuals, even in defined environments.
Prior art observations suggest that up-regulation of the mammalian gene (odc) encoding ornithine decarboxylase is associated with enhanced susceptibility to carcinogenesis (Luk et al., 1984, N. Engl. J. Med. 311:80-83). Other prior art references indicate that overexpression of the odc gene increases susceptibility of mammalian cells for the tumor promotion stage of carcinogenesis, but that over-expression of odc is not, by itself, a sufficient condition for carcinogenesis (Clifford et al., 1995, Cancer Res. 55:1680-1686; Auvinen et al., 1992, Nature 360:355-358; Moshier et al., 1993, Cancer Res. 53:2618-2622; Hibshoosh et al., 1991, Oncogene 6:739-743; O'Brien et al, 1997, Cancer Res. 57:2630-2637).
It is known that there are at least two alleles of the human odc gene, which have been observed as different PstI restriction fragment length polymorphisms (RFLP; Hickok et al., 1987, DNA 6:179-187).
It is believed that the odc gene product, ornithine decarboxylase, is involved in establishing cellular polyamine levels, and that the susceptibility of a tissue to carcinogenesis is related to polyamine levels in the cells of the tissue. Transcription of the odc gene is activated by the protein designated Myc (Bello-Fernandez et al., 1993, Proc. Natl. Acad. Sci. USA 90:7804-7808; Tobias et al., 1995, Oncogene 11:1721-1727; Wagner et al., 1993, Cell Growth Diff. 4:789-883). Such regulation is effected by binding of Myc/Max heterodimers to one or more of the three Myc binding elements (also called “E-boxes”) in the human odc gene. These E-boxes are located at nucleotide residues −485 to −480 (relative to the transcription start site) in the odc 5′-promoter region and at nucleotide residues +292 to +297 and +326 to +331 in the first intron of the odc gene. The fact that the E-box located in the promoter region of the human gene is not conserved in the rat and mouse genes, suggests that the other two E-boxes are the physiologically important regulatory elements.
In normal cells, expression of c-myc (the gene encoding Myc protein) is associated with cell cycle progression. Lack of c-myc expression permits cells to withdraw from the cell cycle and cease differentiating. Furthermore, constitutive c-myc expression promotes continuous cell cycle progression independent of growth factors (Rapp et al., 1985, Nature 317:434-438). Over-expression of c-myc is a common characteristic of many (if not most) human tumors. Prior art studies have correlated c-myc expression and odc expression in human tumors, but not in surrounding normal (i.e. non-tumor) tissue (Gan et al., 1993, J. Histochem. Cytochem. 41:1185-1196; Mori et al., 1996, Cancer 77{8 Suppl.}:1634:1638; Mimori et al., 1997, Dis. Colon Rectum 40:1095-1100). These studies suggest that deregulation of c-myc expression may lead to deregulation of odc expression.
Numerous stimuli are known which induce deregulation of c-myc expression (Schwab et al., 1984, Proc. Natl. Acad. Sci. USA 81:4940-4944; Payne et al., 1982, Nature 295:209-214; Klein et al., 1985 Immunol. Today 6:208-215). Because deregulated Myc transactivates odc expression, these stimuli can be predicted to induce deregulation of odc expression in general. However, until the present invention, it has not been known whether either of the alleles of odc is more susceptible to transactivation by Myc, and thus the genetic component of carcinogenic susceptibility of an individual could not be ascertained.
BRIEF SUMMARY OF THE INVENTION
The invention relates to a method of assessing the relative susceptibility of a mammal (e.g. a human) to an epithelial cancer. The epithelial cancer can, for example be a skin cancer (e.g. a squamous cell carcinoma), a cancer of the digestive system, an esophageal cancer, a gastric cancers, a colon cancer, a prostate cancer, a breast cancer, an hematopoietic cancer, a lung cancer, a melanoma, or a cervical cancer. The method comprises determining whether the mammal comprises an A-allele of the odc gene. If the mammal comprises the A-allele, then the mammal has a greater susceptibility to the epithelial cancer than a mammal of the same type which does not comprise the A-allele.
In one embodiment, determining whether the mammal comprises an A-allele of the odc gene comprises amplifying a reference portion of the mammal's genome. By way of example, the reference portion can be amplified using a pair of primers having nucleotide sequences SEQ ID NO: 3 and SEQ ID NO: 4. Optionally, the reference portion can be further amplified using a pair of nested primers having nucleotide sequences SEQ ID NO: 5 and SEQ ID NO: 6. The reference portion preferably comprises a region (e.g. intron 1) of the odc gene, such as nucleotide residues +282 to +340 relative to the transcription start site. The reference portion can, for example, have a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. The region preferably comprises the nucleotide residue located at position +321 relative to the transcription start site.
An oligonucleotide probe can be annealed with a target portion of the mammal's genome prior to amplifying the reference portion. The target portion includes the nucleotide residue located at position +321 relative to the transcription start site of the odc gene. The probe can, optionally, comprise a fluorescent label such as FAM, TET, rhodamine, VIC, JOE, or Hex. The probe can further comprise a fluorescence quencher such as TAMRA or DABCYL. For example, the probe can have the nucleotide sequence SEQ ID NO: 14 and comprise both a fluorescent label and a fluorescent quencher When the probe comprises both the label and the quencher, one is preferably attached to the probe within 10 nucleotide residues of the 3′-end of the probe and the other is preferably attached to the probe within 10 nucleotide residues of the 5′-end of the probe. The reference portion can be amplified using a DNA polymerase having 5′→3′ exonuclease activity, such as
Thermus aquaticus
DNA polymerase. Preferably, the probe has a length from 15 to about 30 nucleotide residues and the size of the reference portion is not more than about 100 nucleotide residues.
In another embodiment of this method of the invention, determining whether the mammal comprises an A-allele of the odc gene comprises contacting a polynucleotide derived from the mammal's genome with a molecular beacon probe. The probe has a targeting portion which is complementary to a target region of the odc gene, and the target region includes the nucleotide residue located at position +321 relative to the transcription start site of the odc gene. Preferably, the targeting portion is completely complementary to the target region of the A-allele of the odc gene and has a length from about 20 to about 40 nucleotide residues. For example, the targeting portion of the first probe can have the nucleotide sequence SEQ ID NO: 14, and the targeting portion of the second probe can have the nucleotide sequence SEQ ID NO: 15. The target region can, for example, comprise about 20 to about 30 consecutive nucleotide residues of a polynucleotide having a sequence selected from the group consisting of SEQ ID NOs: 1 and 2. The polynucleotide can, for example
Guo Yong Jun
O'Brien Thomas G.
Akin Gump Strauss Hauer & Feld L.L.P.
Forman B. J.
Jones W. Gary
Lankenau Medical Research Center
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