WO2004003203A1

WO2004003203A1 - Human cervical cancer 5 protooncogene and protein encoded therein

Info

Publication number: WO2004003203A1
Application number: PCT/KR2002/001247
Authority: WO
Inventors: Jin-Woo Kim
Original assignee: Jin-Woo Kim
Priority date: 2002-06-29
Filing date: 2002-06-29
Publication date: 2004-01-08
Also published as: AU2002345407A1

Abstract

The present invention relates to a novel protooncogene Human Cervical Cancer 5 (HCC-5) that is non-homologous to existing oncogenes and the protein encoded by the oncogene. This novel protooncogene can be advantageously used in diagnosing various cancers; in construction of transformed animals; and in anti-sense gene therapy.

Description

HUMAN CERVICAL CANCER 5 PROTOONCOGENE AND PROTEIN ENCODED THEREIN

Field of the Invention

The present invention relates to a novel protooncogene and protein encoded therein, and more particularly, to a human cervical cancer 5 protooncogene and a protein derived therefrom, which can be used in diagnosis of various cancers.

Background of the Invention

Higher animals including man each carry approximately 100,000 genes, but only about 15 % thereof is expressed, and characteristics of individual's biological process, e.g., genesis, differentiation, homeostasis, responses to stimuli, control of cell segmentation, aging and apoptosis (programmed cell death), are determined depending on which genes are expressed (Liang, P. and A. B. Pardee, Science, 257: 967-971(1992)).

Pathogenic phenomena such as tumorigenesis are caused by gene mutation, which brings about changes in the mode of gene expression.

Therefore, comparative studies of gene expressions in various cells have been conducted to provide bases for establishing viable approaches to the understanding of diverse biological phenomena.

For example, the mRNA differential display (DD) method suggested by Liang and Pardee is effective in elucidating the nature of tumor suppressor genes, cell cycle-related genes and transcriptional regulatory genes that control apoptosis (Liang, P. and A. B. Pardee supra). Further, the DD method has been widely used in examining the interrelationship of various genes in a cell.

It has been reported that tumorigenesis is caused by various genetic changes such as the loss of chromosomal heterozygosity, activation of oncogenes and inactivation of tumor suppressor genes, e.g., p53 gene (Bishop, J. M., Cell, 64: 235-248(1991); and Hunter, T., Cell, 64: 249-270(1991)). Further, it has been reported that 10 to 30% of human cancer arises from the activation of oncogenes through amplification of protooncogenes.

Therefore, the activation of protooncogenes plays an important role in the etiology of many tumors and there has existed a need to identify protooncogenes.

The present inventor has endeavored to unravel the mechanism involved in the tumorigenesis of cervical cancer; and, has unexpectedly found that a novel protooncogene, human cervical cancer 5 (HCC-5), is specifically overexpressed in cancer cells. This protooncogene can be advantageously used in diagnosis, prevention and treatment of various cancers, e.g., leukemia, lymphoma, skin, colon, breast, kidney, stomach, lung, ovary and uterine cervix cancers.

Summary of the Invention

Accordingly, the primary object of the present invention is to provide a novel protooncogene and a fragment thereof.

Other objects of the present invention are to provide: a recombinant vector containing said protooncogene or a fragment thereof and a microorganism transformed therewith; a protein encoded in said protooncogene and a fragment threrof; a kit for diagnosis of cancer containing said protooncogene or a fragment thereof; a kit for diagnosis of cancer containing said protein or a fragment thereof; an anti-sense gene having a base sequence complementary to that of said protooncogene or a fragment thereof; and a process for treating or preventing cancer by using said anti-sense gene. In accordance with one aspect of the present invention, there is provided a novel protooncogene having the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof.

In accordance with another aspect of the present invention, there is provided a recombinant vector containing said protooncogene or a fragment thereof and a microorganism transformed with said vector.

In accordance with still another aspect of the present invention, there is provided a protein having the amino acid sequence of SEQ ID NO: 2 or a fragment thereof derived from said protooncogene or a fragment thereof.

Brief Description of the Drawings

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings which respectively show;

Fig. 1 : the result of DDRT-PCR for CG262 expressed in normal cervix tissue, primary cervical cancer tissue, metastatic lymph node tissue and CUMC- 6 cervical cancer cells.

Fig. 2A: the results of northern blot analyses for HCC-5 gene expressed in normal cervical tissues, primary cervical cancer tissues and cervical cancer cell lines CUMC-6 and CaSki.

Fig. 2B: the results obtained with the same sample of Fig. 2 A hybridized with β-actin;

Fig. 3 A: the results of northern blot analyses for HCC-5 gene expressed in normal human 12-lane multiple tissues; Fig. 3B: the results obtained with the same sample of Fig. 3 A hybridized with β-actin;

Fig. 4A: the results of northern blot analyses for HCC-5 gene expressed in human cancer cell lines;

Fig. 4B: the results obtained with the same sample of Fig. 4A hybridized with β-actin; and

Fig. 5: sodium dodecyl sulfate (SDS)-PAGE results showing protein expression patterns and protein size before and after the IPTG induction.

Detailed Description of the Invention

The novel protooncogene of the present invention, i.e., human cervical cancer 5 (hereinafter "HCC-5 protooncogene"), consists of 3,091 base pairs and has the DNA sequence of SEQ ID NO: 1.

In SEQ ID NO: 1, the full open reading frame corresponding to base Nos. 54 to 2,084 (2,082-2,084: temiination codon) is a protein encoding region and the predicted amino acid sequence derived therefrom is shown in SEQ ID NO: 2 which consists of 676 amino acids (hereinafter "HCC-5 protein").

In consideration of the degeneracy of codons and the preferred codons in a specific organism wherein the protooncogene of the present invention is to be expressed, various changes and modifications of the DNA sequences of SEQ ID NO: 1 may be made, e.g., in the coding area thereof without adversely altering the amino acid sequence of the expressed protein, or in the non-coding area without adversely affecting the expression of the protooncogene. Therefore, the present invention also includes, in its scope, a polynucleotide having substantially the same base sequence as the inventive protooncogene, and a fragment thereof. As used herein, "substantially the same polynucleotide" refers to a polynucleotide whose base sequence shows 80% or more, preferably 90% or more, most preferably 95% or more homology to the protooncogene of the present invention.

The protein expressed from the protooncogene of the present invention consists of 676 amino acids and has the amino acid sequence of SEQ ID NO: 2. The molecular weight of this protein is about 76 kDa. However, various substitution, addition and/or deletion of the amino acid residues of protein may be performed without adversely affecting the protein's function. Further, a portion of the protein may be used when a specific purpose is to be fulfilled. These modified amino acids and fragments thereof are also included in the scope of the present invention. Therefore, the present invention includes, in its scope, a polypeptide having substantially the same amino acid sequence as the protein derived from the oncogene of the present invention and a fragment thereof. As used herein, "substantially the same polypeptide" refers to a polypeptide whose amino acid sequence shows 80 % or more, preferably 90 % or more, most preferably 95 % or more homology to the amino acid sequence of SEQ ID NO: 2.

The protooncogene or the protein of the present invention can be obtained from human cancer tissues or synthesized using a conventional DNA or peptide synthesis method. Further, the gene thus prepared may be inserted to a conventional vector to obtain an expression vector, which may, in turn, be introduced into a suitable host, e.g., a microorganism such as an E. coli or yeast, or an animal cell such as a mouse or human cell. The cells transformed with a vector containing the HCC-5 protooncogene or a fragment thereof is hereinafter referred to a "HCC-5 cell". The transformed host may then be used in producing the inventive DNA or protein on a large scale. For example, E. coli DH5α was transfected with an expression vector comprising protooncogene HCC-5, and E. coli DH5α/HCC-5/ρCEV-LAC thus obtained was deposited on April 10, 2001 with the Korean Collection for Type Cultures (KCTC) (Address: Korea Research Institute of Bioscience and Biotechnology (KRIBB), #52, Oun-dong, Yusong- ku, Taejon, 305-333, Republic of Korea) under the accession number of KCTC 0988BP, in accordance with the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure. In preparing a vector, expression-control sequences, e.g., promoter, terminator, self-replication sequence and secretion signal, are suitably selected depending on the host cell used.

The overexpression of the protooncogene of the present invention occurs not in normal cervical and lung tissues but in cervical cancer tissues and cervical cancer cell lines. This suggests that the protooncogene of the present invention induces cervical cancers. In addition to epithelial tissues such as cervical cancer tissue, the over expression of the protooncogene of the present invention is also observed in various other cancer tumors such as leukemia, lymphoma, breast, kidney, ovary, lung and stomach cancers. Therefore, the protooncogene of the present invention is believed to be a factor common to all forms of cancers and it can be advantageously used in the diagnosis of various cancers and the production of a transformed animal as well as in an anti-sense gene therapy.

A diagnostic method that can be performed using the protooncogene of the present invention may comprise, for example, the steps of hybridizing nucleic acids separated from the body fluid of a subject with a probe containing the protooncogene of the present invention or a fragment thereof, and determining whether the subject has the protooncogene by using a conventional detection method known in the art. The presence of proto-oncogene may be easily detected by labeling the probe with a radioisotope or an enzyme. Therefore, a cancer diagnostic kit containing the proto-oncogene of the present invention or a fragment thereof is also included in the scope of the present invention.

A transformed animal produced by introducing the proto-oncogene of the present invention into a mammal, e.g., mice, is also included in the scope of the present invention. In producing such a transformed animal, it is preferred to introduce the inventive protooncogene to a fertilized egg of an animal before the 8-cell stage. The transformed animal can be advantageously used in screening for carcinogens or anticancer agents such as chemotherapeutic drugs.

The present invention also provides an anti-sense gene, which is useful in a gene therapy. As used herein, the term "an anti-sense gene" means a polynucleotide comprising a base sequence which is fully or partially complementary to the sequence of the mRNA which is transcribed from the protooncogene having the base sequence of SEQ ID NO: 1 or a fragment thereof, said nucleotide being capable of preventing the expression of the open reading frame (ORF) of the protooncogene by way of attaching itself to the protein-binding site of mRNA. The present invention also includes within its scope a process for treating or preventing cancer in a subject by way of administering a therapeutically effective amount of the inventive anti-sense gene thereto.

In the inventive anti-sense gene therapy, the anti-sense gene of the present invention is administered to a subject in a conventional manner to prevent the expression of the protooncogene. For example, the anti-sense HCC-5 is mixed with a hydrophobicized poly-L-lysine derivative by electrostatic interaction in accordance with the method disclosed by Kim, J.S. et al. (J. Controlled Release, 53: 175-182(1998)) and the resulting mixed anti- sense oligodeoxynucleotide (ODN) is administered intravenously to a subject.

The present invention also includes within its scope an anti-cancer composition comprising the anti-sense gene of the present invention as an active ingredient, in association with pharmaceutically acceptable carriers, excipients or other additives, if necessary. The pharmaceutical composition of the present invention is preferably formulated for administration by injection.

The amount of the anti-sense gene actually administered should be determined in light of various relevant factors including the condition to be treated, the chosen route of administration, the age and weight of the individual patient, and the severity of the patient's symptoms. The protein expressed from the inventive protooncogene may be used in producing an antibody useful as a diagnostic tool. The antibody of the present invention may be prepared in the form of a monoclonal or polyclonal antibody in accordance with any of the methods well known in the art by using a protein having the amino acid sequence of SEQ ID NO: 2 or a fragment thereof. Cancer diagnosis may be carried out using any of the methods known in the art, e.g., enzyme linked immunosorbentassay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemical staining, western blot or immunoassay blot on polyacrylic gel, to assess whether the protein is expressed in the body fluid of the subject. Therefore, a cancer diagnostic kit containing the protein having the amino acid sequence of SEQ ID NO: 2 or a fragment thereof is also included in the scope of the present invention. A continuously viable cancer cell line may be established by using the proto-oncogene of the present invention, and such a cell line may be obtained, for example, from tumor tissues formed on the back of a nude mouse by injecting fibroblast cells transformed with the protooncogene of the present invention. The cell lines thus prepared may be advantageously used in searching for anti-cancer agents.

The following Examples are given for the purpose of illustration only, and are not intended to limit the scope of the invention.

Example 1 : Cultivation of tumor cells and isolation of total RNA

(Step 1) Cultivation of tumor Cells

For differential display of mRNA, normal cervical tissues, untreated primary cervical cancer tissues and metastatic common iliac lymph node tissues were obtained from cervical cancer patients who underwent radical hysterectomy. The human cervical cancer cell line used in the differential display method was CUMC-6 cell line described by Kim et al, (Gynecol. Oncol, 62: 230-240(1996)).

The cells obtained from the above-described tissues and CUMC-6 were maintained on Waymouth's MB 752/1 medium (Gibco) supplemented with 2 mmol/L of glutamine, 100 IU/ml of penicillin, 100 μg/ml of streptomycin, and 10 % of fetal bovine serum (Gibco). Only the cell suspensions with greater than 95%» viability, as assessed by trypan blue dye exclusion described by FreshneyC'Culture of Animal Cells: A Manual of Basic Technique" 2nd Ed., A. R. Liss, New York(1987)) were used in the present experiments.

(Step 2) Isolation of RNA and mRNA Differential Display

Total RNAs were extracted from normal cervical tissues, primary cervical cancer tissues, metastatic common iliac lymph node tissues and CUMC-6 cells obtained in Step 1 using a commercial system (RNeasy total

RNA kit) provided by Qiagen (Qiagen Inc., Germany) and the removal of DNA contaminants from the RNAs was accomplished using Message clean kit (GenHunter Corp., Brookline, MA).

Example 2: Differential Display Reverse Transcription (DDRT)-PCR

Differential display reverse transcription was performed in accordance with the reverse transcription-polymerase chain reaction (RT-PCR) method described by Liang and Pardee(1992), supra, with minor modifications.

0.2 μg each of the total RNAs obtained in Step 1 of Example 1 was subjected to reverse transcription using H-Tl IG primer of SEQ ID NO: 3 as an anchored oligo-dT primer (RNAimage kit, GenHunter, cor., MA, USA), followed by polymerase chain reaction (PCR) using the same anchored primer and the primer of SEQ ID NO: 4 (H-AP26 primer among RNAimage primer sets 1-4, H-AP 1-32) in the presence of 0.5 mM [α -³⁵S]-labeled dATP (1200 Ci/mmol). The PCR thermal cycle was repeated 40 times, each cycle being composed of: 95 °C for 40 sec, 40 °C for 2 min. and 72 ^°C for 40 sec, and the final extension reaction was carried out at 72 °C for 5 min. The PCR product thus obtained was subjected to electrophoresis in 6 % polyacrylamide sequencing gels, followed by autoradiography to determine the location of bands expressed differentially.

The band of fragment CG262 cDNA of size 292 bp (sequence 2,679- 2,970 bp) was excised from the dried sequencing gel and then heated for 15 min. to elute fragment CG262 cDNA. The fragment CG262 cDNA was amplified o r by conducting PCR under the same conditions except that [α - S]-labeled dATP and 20 μM dNTPs were omitted.

Example 3 : Cloning

The amplified fragment CG262 was cloned into pGEM-T Easy vector using the TA Cloning System (Promega, USA). (Step 1) Ligation Reaction

2 μl of CG262 product obtained in Example 2, 1 μl ofpGEM-T Easy vector (50 ng), 1 μl of T4 DNA ligase 10 X buffer solution, and 1 μl of T4

DNA ligase (3 weiss unit/μβ; T4 DNA ligase, Promega) were added into a 0.5 mi test tube. Then, distilled water was added to a total volume of 10 μl and cultured at 14 ^°C over night.

(Step 2) TA Cloning Transformation

TA cloning transformation was conducted as follows: E. Coli JM109 was cultured in 10 mi LB medium (Bacto-Trip 10 g,

Bacto-yeast extract 5 g, NaCl 5 g) until its optical density reached approximately 0.3 to 0.6 at 600 nm. The culture mixture was kept in ice for about 10 minutes, then centrifuged at 4 ^°C for 10 minutes at 4,000 rpm in order to isolate bacterial cells. The bacterial cells thus obtained were exposed in 10 mi of 0.1 M CaCl₂ for 30 minutes to 1 hour to produce competent cells. The resultant mixture was centrifuged at 4 ^°C for 10 minutes at 4,000 rpm. The cells are collected and suspended in 2 ml 0.1 M CaCl₂.

The competent cell suspension was placed in a new 200 μl microfuge tube, then 2 μl of the ligation solution obtained in step 1 was added thereto. The mixture was cultured in a 42 °C water bath for 90 seconds, and then, chilled quickly to 0 ^°C . 800 μl of SOC medium (Bacto-Tripton 2.0 g, Bacto-yeast extract 0.5 g, 1 M NaCl 1 m!, l M KC1 0.25 ml, TDW 97 mi, 2 M Mg²⁺, 2 M Glucose 1 mi) was added thereto, and the mixture was incubated at 37 ^°C in a rotary shaking incubator at 220 rpm for 45 minutes. 25 μl of X-gal (stored in 40 mg/m^ dimethylformamide) was smeared onto an ampicillin-containing LB plate stored in a 37 ^°C incubator using a glass rod. Then 25 μl of the transformed cells were smeared onto said plate using a glass rod, and incubated overnight at 37 ^°C . 3 to 4 colonies were selected from the plate and cultured on separate ampicillin-containing LB plates. In order to produce a plasmid, a transformed E. Coli JM109/CG262 was selected and cultured on 10 mi of terrific broth (TDW 900 ml, Bacto-Trip 12 g, Bactor-yeast extract 24 g, glycerol 4 ml, 0.17 M KH₂P0₄, 0.72 M K₂HP0₄ 100 mi).

Example 4: Separation of Recombinant Plasmid DNA

Using the Wizard™ Plus Minipreps DNA Purification Kit (Promega, USA), CG262 plasmid DNA was separated from the transformed E. Coli.

The separated plasmid DNA was treated with EcoRI restriction enzyme, and subjected to 2 % gel electrophoresis to confirm the insertion of CG262 sequence in the plasmid.

Example 5: Analysis of DNA base sequence

The CG262 PCR product obtained in Example 2 was amplified using a known method, and cloned. The sequence of the amplified CG262 PCR fragment, analyzed using the Sequenase version 2.0 DNA sequencing kit

(United States Biochemical, Cleveland, OH, USA) according to the dideoxy chain termination method, corresponded to nucleotide numbers 2,679 to 2,970 of SEQ ID NO: 1 and this DNA fragment was designated "CG262". Using the 3 ' H-Tl 1 G primer of SEQ ID NO: 3 and 5 ' random primer H-

AP26 of SEQ ID NO: 4, the cDNA fragment of 292 bp obtained above

(CG262) was subjected to DDRT-PCR and then verified through electrophoresis. It was found that the mode of gene expression of this fragment varied depending on the kind of tissue: As illustrated in Fig. 1, the cDNA fragment (CG262) was expressed in the cervical cancer, metastatic tissues and CUMC-6 cervical cancer cells but not in normal tissues.

Example 6: Full length cDNA sequence analysis of the HCC-5 Protooncogene

A bacteriophage λgtll human lung embryonic fibroblast cDNA library

(Mild, T. et. al., Gene, S3: 137-146(1989)) was screened by plaque hybridization with ³²P-labeled CG262 cDNA probe. The full-length HCC-5 cDNA clone, containing a 3,105 bp insert in vector pCEV-LAC was obtained from the human lung embryonic fibroblast cDNA library.

HCC-5 clone inserted into λpCEV vector (see Mild, T. et al., supra) was excised out of the phage in the form of the ampicilline-resistant pCEV-LAC phagemid vector (see Mild, T. et al., supra) by Notl cleavage.

To make a HCC-5 plasmid DΝA, pCEV-LAC vector containing HCC-5 gene was ligated with T4 DΝA ligase and ligated clone was transformed into E. coli DH5α. The transformed E. Coli DH5α/HCC-5/pCEV-LAC thus obtained was deposited on April 10, 2001 with Korean Collection for Type Cultures (KCTC) (Address: Korea Research Institute of Bioscience and Biotechnology (KRIBB), #52, Oun-dong, Yusong-ku, Taejon, 305-333, Republic of Korea) under the accession number, KCTC 0988BP, in accordance with the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure.

The full sequence of HCC-5 consists of 3,105 bp which is identified in SEQ ID NO: 1.

In SEQ ID NO: 1, the full open reading frame of the HCC-5 protooncogene of the present invention corresponds to nucleotides No. 54 to 2,084 and is predicted to encode amino acid sequence shown in SEQ ID NO: 2 which consists of 676 amino acids.

Example 7: Northern blot analysis of the HCC-5 gene in various cells

Total RNAs were extracted from various tissues and cell lines as in Example 1.

To determine the level of HCC-5 gene expression, 20 μg denatured total

RNAs from each tissue or cell lines were electrophoresed through 1 % formaldehyde agarose gel and transferred to nylon membranes (Boehringer-

Mannheim, Germany). The blots were hybridized with a ³²P-labeled random- primed HCC-5 full cDNA probe which was prepared using a Rediprime II random prime labeling system (Amersham, England). The northern blot analysis was repeated twice and the results were quantified by densitometry and normalized with β-actin. Fig. 2A shows the results of northern blot analyses for HCC-5 gene expressed in normal cervical tissues, primary cervical cancer tissues and cervical cancer cell lines CUMC-6 and CaSki. Fig. 2B shows the results obtained with the same samples hybridized with a β-actin probe to confirm mRNA integrity. As can be seen from Fig. 2, the expression level of HCC-5 gene was elevated in the cervical cancer tissues and the cervical cancer cell lines but nearly absent in all normal cervical tissues.

Fig. 3A shows the results of northern blot analyses for HCC-5 gene expressed in normal human 12-lane multiple tissues; brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and leukocyte tissues(Clontech). Fig. 3B shows the results obtained with the same samples hybridized with a β-actin probe to confirm mRNA integrity. As can be seen in Fig. 3 A, HCC-5 mRNA (-3.1 kb) is faint or nearly absent in normal tissues.

Fig. 4A shows the results of northern blot analyses for HCC-5 gene expressed in human cancer cell lines; HL-60, HeLa, K-562, MOLT-4, Raji, SW480, A549 and G361(Clontech). Fig. 4B shows the results obtained with the same samples hybridized with a β-actin probe to confirm mRNA integrity. As can be seen in Fig. 4A, HCC-5 is transcribed especially at a high level in lymphoblastic leukemia MOLT-4, and is overexpressed in Burkitt's lymphoma Raji, skin cancer cell line G361, promyelocytic leukemia HL-60, HeLa uterine cancer cell line, chronic myelogenous leukemia K-562, SW480 colon cancer cell, and lung cancer cell line A549.

Example 8: Determination of the size of the protein expressed after the transfection of E. Coli with HCC-5 protooncogene A full-length HCC-5 protooncogene of SEQ ID NO: 1 was inserted into the multiple cloning site of pGEX-4T-3 vector (Amersham Pharmacia) and the resulting pGEX-4T-3/HCC-5 vector was transfected into E. coli BL21 (ATCC 47092). Glutathione-GST-transferase is inserted at the front of the pGEX-4T- 3 vector multiple cloning site. The transfected E. coli was incubated using an LB broth medium in a rotary shaking incubator, diluted by 1/100, and incubated for 3 hours. 1 mM isopropyl β-D-thiogalacto-pyranoside (IPTG, Sigma) was added thereto to induce the protein synthesis.

The E. coli cells in the culture were disrupted by sonication and subjected to gel electrophoresis using 12 % sodium dodecyl sulfate (SDS) before and after the IPTG induction. Fig. 5 shows the SDS-PAGE results, which exhibit a protein expression pattern of the E. coli BL21 strain, transfected with ρGEX-4T-3/HCC-5 vector. After the IPTG induction, a significant protein band was observed at about 102 kDa. This 102 kDa fused protein contained GST protein of about 26 kDa and HCC-5 protein of approximately 76 kDa.

III IDΛIT.!IT TREATY oκ TUP. INTP.HNATIUN-AI. IU- OUKITION OK TUB UEIΌSIT

OP MICHOOIICΛNIK H Will TI IK I'tllll'OSli OF PATBN'Ϊ PHOCEIΛJRE

INTERNATIONAL PORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Hule 7.1

: KIM, Jin Woo l lyuπdaK Apt. 1 IH 804, Λpgujung-dong, angπam-gu, Seoul 135-1 10. ltepublic of Korea

1 . IDENTIFICATION OF THE MICROORGANISM

Accession number given by the

Identification reference given by the INTERNATIONAL DEPOSITARY DEPOSITOR: AUTHORITY-' πacherichia coli KCTC 0988BP DH5@/HCC-5/»CEV~LAC

LI . SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONO IC DEStfiNATfON

The microorganism identified under I abov was accompanied b : r x 1 a scientific description

L 1 a proposed taxonαmic designation

(Mark with a cross where applicable)

HI. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the miciTiortfaniirπ identified under I above, which ns received by it on April 10 2001.

IV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this International Depositary Authority on and a request to convert (he original deposit to a deposit, under the Budapest Treaty was received by it on

V. INTERNATIONA I , DEPOSITARY APTHOKITY

Name: Korean Collection for Type Cultures Siκnature(s) of person (s) having the power to represent the International Depositary Authority of authorized offidaKs):

Address: Kor Research Institute of Biospttrure arid Biotechnology (KRIBB⁾

# , Ourv-dong, Yuaoπg -leu, Taejnn 308-333, BAE, Kyuriβ Soak, Director Republic of Korea Date: April 16 2001

Claims

What is claimed is :

1. A human cervical cancer protooncogene having the base sequence of SEQ ID NO: 1 or a fragment thereof.

2. The fragment of the protooncogene of claim 1 having a base sequence corresponding to base Nos. 54 to 2,084 of SEQ ID NO: 1.

3. A protein having the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.

4. A vector comprising the protooncogene or fragment of claim 1.

5. A microorganism transformed with the vector of claim 4.

6. The microorganism of claim 5, which is E. coli DH5α/HCC-5/pCEV-LAC (Accession No.: KCTC 0988BP).

7. A process for preparing the protein or fragment of claim 3 comprising the step of culturing the microorganism of claim 5 or 6.

8. A kit for diagnosis of cancer, which comprises the protooncogene or fragment of claim 1 or 2.

9. A kit for diagnosis of cancer, which comprises the protein or fragment of claim 3.

10. An anti-sense gene having a base sequence which is complementary to the sequence of the full or partial mRNA transcribed from the protooncogene or fragment of claim 1 or 2 and being capable of binding the mRNA to inhibit the expression of said protooncogene or fragment.

1. A composition for treating or preventing cancer, which comprises a therapeutically effective amount of the anti-sense gene of claim 10 and a pharmaceutically acceptable carrier.