KR100400987B1 - Novel Oncogene of Mouse - Google Patents

Abstract

The present invention relates to a polypeptide expressed from a mouse oncogene and an electric gene comprising a nucleotide sequence having high homology with the nucleotide sequence of HCCR1, which is one of the causative genes for cervical cancer in human women. MCCR, a mouse cancer gene of the present invention, has a high homology in terms of nucleotide sequence and amino acid sequence expressed by a gene, compared with HCCR1, which is a human cervical cancer onset gene, and has a similar expression mechanism. It can be usefully used as an experimental model to study.

Description

Cancer gene of mouse {Novel Oncogene of Mouse}

The present invention relates to oncogenes of mice. More specifically, the present invention relates to a polypeptide expressed from a mouse oncogene and an electric gene comprising a nucleotide sequence having a high homology with the nucleotide sequence of HCCR1, which is one of the causative genes for cervical cancer in women.

The incidence and mortality rate of cervical cancer has recently decreased due to improved diagnostic methods, but it still accounts for 23% of malignant tumors in Korean women. Number one among cancers, making it a major public health concern (Lund, R., Obstet Gynecol., 63: 708-713, 1984; Ministry of Health and Welfare, Analysis of Korean Cancer Registry Survey Data, 1995). Moreover, cervical cancer has begun to change and the incidence has increased in young women in the mid-30s since the 1980s, and in cases of cervical cancer in young women, the histological differentiation is more malignant and lymph node metastases. Findings are also common, and the survival rate of patients has been reported to be very low (see Eliott, PM et al., Brit. Med. J., 298: 288-290, (1989)).

Currently, human papillomavirus (hereinafter referred to as HPV) plays an important role in cervical cancer, but carcinogenic HPV is detected in normal women. This is a transition to cervical cancer (zur Hausen, H., Cancer Res., 49: 4677-4681 (1989)). HPV infection is not always accompanied by malignant changes of cervical epithelial tissues. In addition, it is estimated that environmental factors, immunological characteristics, and genetic factors work together to develop cervical cancer.

When the human body is exposed to various chemical carcinogens, the characteristics and degree of exposure of the carcinogen itself also play an important role in the development of a particular cancer, but also greatly influenced by the difference in genetic susceptibility of each individual. (Idle, JR, Mutat. Res., 247: 259-266, 1991; Nebert, DW, Mutat. Res., 247: 267-281, 1991). In recent years, researches on the effects of genetic sensitivity on individuals have been actively conducted on the onset of various cancers, which are closely related to smoking. Genetic polymorphism analysis of various enzymes involved in the metabolism of procarcinogen has been attracting attention as a method for analyzing the influence of genetic susceptibility between individuals in the carcinogenic process. Examples include enzymes involved in drug metabolism, such as isoenzymes of cytochrome P450 (CYP), CYP 1A1 and CYP 2E1, and glutathione S-transferase (GST). .

On the other hand, polymorphism analysis of the enzyme gene can be easily performed by combining the polymerase chain reaction (PCR) and restriction enzyme fragment length polymorphism (RFLP) analysis. First, the CYP 1A1 gene is located on human chromosome 15. The polymorphism of the electric gene was first studied and found in Japanese (see Hildebrand, CE et al., Nucleic Acids Res., 113: 2009-2016, 1985). Kawajiri, K. et al., FEBS Lett., 236: 131-133, 1990). The distribution of Msp I polymorphism of CYP1A1 gene in Japanese is 69% wild type and 31% variant type, especially in lung cancer patients with homozygous variant. Even more commonly found (Hashiashi, SI et al., J. Biochem., 110: 559-565, 1991; Hayashi, SI et al., Jpn. J. Cancer Res., 83: 866-870, 1992 ). Next, the CYP 2E1 gene is a phase I drug metabolic enzyme, which occurs not only in smoking but also in various environments including food, and is known to cause cancer in the human body. It acts on the metabolic processes of various chemical pro-carcinogens such as nitrosamine, aniline, benzene, vinyl chloride, urethane, etc., and converts them to activated carcinogens. (Guengerich, FP and Shimada, T. Chem. Res. Toxicol., 4: 391-407, 1991). To date, Pst I or Rsa I polymorphisms located in the 5'-flanking region of the CYP2E1 gene and Dra I polymorphisms located in intron 6 are known. Among these, specific genotypes include digestion such as liver cancer. It has been reported to be associated with the development of mechanical cancer (Uematsu, F. et al., Jpn. J. Cancer Res., 82: 254-256, 1991; Hayashi, SI, J. Biochem., 110: 407-411, 1991; Uematsu, F. et al., Pharmocogenetics, 4: 58-63, 1994; Yu, MW et al., Gastroenterology, 109: 1266-1273, 1995).

In addition, GST is a phase II metabolic enzyme, which catalyzes the reduced glutathione synthesis reaction of various activated developmental carcinogens and decodes the electric developmental carcinogens. (Mu), alpha, Pi, and theta classes, of which polymorphisms in GSTM1 in the Mu class and GSTT1 in theta class have been reported to play a large role in determining genetic susceptibility. See Mannervik, B. and Danielson, UH, Crit. Rev. Biochem.Mol. Biol., 23: 281-337, 1988; Bell, DA et al., J. Natl. Cancer Inst., 85: 1159-1164 , 1993). GST enzymes play an important role in detoxifying the activated metabolites of benzo (α) pyrene, a deficiency of which is known to pose a high risk of cancer associated with smoking. Idle, JR, Mutat Res., 247: 259-266, 1991). In addition to the polymorphisms of the electrical enzyme gene, the p53 wiring (germ line) polymorphism i.e., exon 4 / Acc II polymorphism and intron 3 / 16bp duplicate (duplication) polymorphisms are also are associated with lung cancer. Recently, exon 4 / Acc II polymorphism It has been reported to be significantly correlated in HPV related cancers (Birgander, R., Carcinogenesis, 16: 2233-2236, 1995; Storey, A., Nature, 393: 229-234, 1998).

At present, research to identify the above-described mechanism of cervical cancer has been conducted, but no progress has been made yet. The main reason for the lack of progress in the study is the lack of an effective experimental model to identify the causes of cervical cancer. Until now, studies have been conducted on mammalian cancer genes similar to humans. However, due to the complex mechanism of the oncogenes, it was not possible to selectively develop cervical cancer, so the pathogenesis of cervical cancer could not be studied properly. .

Therefore, there is a constant need to develop an experimental model that can selectively study cervical cancer and to effectively study the pathogenesis of cervical cancer.

Accordingly, the present inventors have made efforts to develop an experimental model that can selectively study only cervical cancer and effectively study the pathogenesis of cervical cancer, and as a result, genes and sequences of human cervical cancer among genetically simple mouse genes have been studied. It was confirmed that the present invention can develop a gene having high homology in terms of amino acid sequence expressed by a gene, and have similar expression mechanisms, and use the electric gene as an experimental model for effectively studying the pathogenesis of cervical cancer. To complete.

After all, an object of the present invention is to provide a cancer gene of a mouse having a nucleotide sequence showing a high homology with the nucleotide sequence of the oncogene that causes human cervical cancer.

Another object of the present invention is to provide an amino acid sequence inferred from the base sequence.

1 is a graph showing the analysis results of polypeptides inferred from the MCCR gene.

Figure 2 is a photograph showing the result of Northern blotting the RNA of each tissue cell with MCCR gene.

MCCR, a mouse cancer gene of the present invention, includes a nucleotide sequence having homology with the nucleotide sequence of HCCR1, a cancer gene that causes human cervical cancer.

Hereinafter, the present invention will be described in detail.

The present inventors searched for 2,447 bp mouse genes homologous to HCCR1, a human cancer gene, from mouse embryonic tumor cell cDNA library, and analyzed their sequencing, indicating that the found gene was a novel gene. there was. The gene was expressed in the heart, kidney, uterus, liver, brain, and lung of the mouse, was expressed in tumor cells rather than normal cells, and analyzed by the amino acid sequence of 360 amino acids derived from the base sequence of the gene. It was found that the part has a hydrophobic transmembrane part consisting of about 20 amino acids, and the N-glycosylation site is present at the amino terminal and the carboxy terminal.

The oncogene of the electric mouse showed about 83% homology with the human HCCR1 gene, and the high homology was found when comparing the protein produced by the expression of HCCR1 with the protein obtained by expressing the oncogene of the mouse. Therefore, it was confirmed that the novel gene obtained in the mouse is a cancer gene having a function similar to that of human HCCR1, and named it 'mouse cervical cancer receptor (MCCR)'.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Screening and Analyzing Homologous Sequences of Cancer Genes in Mice

Screening mouse positive mouse cDNA library (Mouse Embryonic Carcinoma cDNA library, Stratagene) using 2,117 bp HCCR1 gene obtained by differential display PCR (dd-PCR) and screening method The clones of the genes were cloned to obtain multiple clones. The clone was infected with λ phage, λ DNA containing a gene of mouse reacted with HCCR1 using λ DNA purification kit (λ DNA purification kit, Qiagen), and then analyzed for their base sequence. It was found to be a partial fragment of the gene. In order to obtain their overall genes, a 5'-terminal amplification method using a 5'-RACE kit (Gibco BRL) was performed on clones having the largest nucleotide sequence among them. After obtaining a gene and confirming its nucleotide sequence (SEQ ID NO: 1), it was named 'MCCR (mouse cervical cancer receptor)'.

As a result of analyzing the homology between the MCCR base sequence and the HCCR1 base sequence, it was found that the homology was about 83%. In addition, the amino acid sequence of the polypeptide consisting of 360 amino acids inferred from the base sequence of the MCCR (SEQ ID NO: 2) was analyzed using the TMPRED program (http://www.ch.embnet.org/software/TREMED_form.html). As a result, as shown in FIG. 1, it has a transmembrane domain consisting of 20 amino acids having strong hydrophobicity in the middle of the polypeptide, and glycosyl at each of the N- and C-terminus. It was assumed that the site would exist. As a result of comparing the homology between the amino acid sequence of MCCR and the amino acid sequence of HCCR1, the homology of 76% was obtained.

As a result, the MCCR gene of the present invention has a nucleotide sequence similar to that of the HCCR1 gene, and since the amino acid sequence of the polypeptide expressed from the aforementioned MCCR gene and the amino acid sequence of the polypeptide expressed from the HCCR1 are similar, the MCCR has a similar function to that of the HCCR1 gene. It was estimated.

Example 2 Expression of MCCR in Mouse Tissues

The obtained MCCR gene was examined in which tissues of the mouse.

First, each RNA was obtained from the spleen, liver, heart, brain, stomach, uterus, kidney, lung and pancreas of the mouse using Trizol reagent (Qiagen), and the MCCR gene obtained in Example 1 was obtained. After labeling with [α-P ³² ] dCTP, Northern blotting was performed using this as a probe, and the expression in each tissue was measured. As a control, 18S rRNA of mouse and 28S rRNA of mouse were measured. Used (see Table 1).

Expression of MCCR in Mouse Tissues Mouse tissue 18S rRNA distance (cm) 28S rRNA distance (cm) MCCR Travel Distance (cm) Zira 8.6 10.2 X Heart 8.1 10.1 8.7 kidney 8.4 10.2 8.9 top 8.4 10.4 X Womb 8.5 10.1 9.1 liver 8.5 10.0 9.0 Pancreas X X X brain 7.7 9.6 8.3 lungs 7.7 9.6 8.3

As shown in Table 1, it can be seen that MCCR is expressed in the heart, kidney, uterus, liver, brain and lung of the mouse, the expression pattern was found to be similar to HCCR1.

Example 3 Expression of MCCR in Cell Lines and Normal Cells

Cell cultures containing 10% FBS (fetal bovine serum) and 1% penicillin from three cancer cell lines derived from mouse muscle cells (Lewis lung carcinoma cell, melanoma cell and T lymphoma cell) and one normal cell (NIH3T3) (Dulbeco's modified Eagle's medium Gibco BRL) was incubated at 37 ^° C with 5% CO ₂ . RNA was then obtained from each of the cultured cells, and Northern blotting was performed by the method of Example 2 to measure the expression level of MCCR in each cell (see FIG. 2). 2 is Northern blotting data of RNA obtained in each cell. In FIG. 2, lane 1 is melanoma cells, lane 2 is T lymphoma cells, lane 3 is Lewis lung carcinoma cells, and lane 4 Is normal cells (NIH3T3). As shown in FIG. 2, since MCCR is expressed in cancer cell lines more than normal cells, it is estimated that MCCR is a cancer gene similar to HCCR1.

As described and demonstrated in detail above, the present invention provides a polypeptide expressed from a mouse oncogene and an electric gene comprising a nucleotide sequence having a high homology with the nucleotide sequence of HCCR1, one of the causative genes for cervical cancer in women. do. MCCR, a mouse cancer gene of the present invention, has a high homology in terms of nucleotide sequence and amino acid sequence expressed by gene, compared with HCCR1, which is a human cervical cancer onset gene, and its expression mechanism is also similar. It can be usefully used as an experimental model to study.

Claims (2)

MCCR gene of a mouse having a nucleotide sequence of SEQ ID NO: 1. A polypeptide having the amino acid sequence of SEQ ID NO: 2 and expressed from the MCCR gene of a mouse.