Patent Application
20160238608
The following application contains a sequence listing in computer readable format (CRF), submitted as a text file in ASCII format entitled “SequenceListing,” created on May 3, 2016, as 22 KB. The content of the CRF is hereby incorporated by reference.
The present invention relates to a human cancer-related gene, its encoded products and their applications in genetic engineering and protein engineering areas, as well as in medical diagnosis and treatment.
Cancer is the major health problem threatening human lives. Hepatocellular carcinoma (HCC) is one of the most serious among cancer diseases. It is reported that the new cases of primary hepatocellular carcinoma exceeds over one million worldwide each year. 70% of the new cases occur in Asia, and about 40-45% of the worldwide new cases occur in China. The total number of new hepatocellular carcinoma cases every year in China is about 450,000, and the number is increasing, especially in those between ages 20-60. The high incidence, difficulty in early diagnosis, fast growing rate, high reoccurrence, and the high mortality rate make HCC a most malignant cancer. Most HCC patients have already progressed to the intermediate stage or late stage when diagnosed, and they can only survive for 3-6 months if without a proper treatment.
To elucidate the mechanism underlying cancerogenesis would help for cancer prevention, diagnosis and treatment. Early diagnosis is crucial for raising the curative rate and reducing the mortality. Currently used HCC-diagnostic marker, the serum AFP, has 30% of negative results in HCC patients, while some benign liver disease can cause a significant increase of AFP level in serum, creating some difficulty in differential diagnosis. It has been found that the hepatocarcinogenesis is related to individual hereditary susceptibility. Individuals with different genetic backgrounds possess different handling capability toward environmental carcinogens. This leads to different risk of suffering from cancer for individuals. It is the various genotypes and the genetic diversity that cause the different genetic susceptibility for cancerogenesis.
Cancer is essentially a cellular hereditary disease. Although a great number of cancer-related genes have been discovered, the mechanisms of the cancerogenesis and the development remain to be elucidated. The known oncogenes can be divided into five categories according to the cellular localization and function of their encoded proteins: I. genes that encode growth factors, including sis, int-2, hst, fgf-5; II. genes that encode growth factor receptors, including erbB, erbB-2, fins, met, ros, and others; III. genes that encode signal transduction molecules in cytoplasm, including abl, src, ras, raf, yes, fgr, fes, lck, mos, and others; IV. genes that encode regulatory molecules for cell proliferation and apoptosis, including bcl-1, bcl-2 and others; and V. genes that encode the nuclear DNA-binding proteins (transcription factors), such as myc, myb, fos, jun, B-lym, ski, ets, rel and others. It has been demonstrated that ras, src, myc, met and p53 etc. are the genes closely associated with HCC.
This invention provides a novel human cancer-related gene and its encoded products.
This novel human cancer-related gene provided by this invention is designated as LAPTM4B. It comprises one of the following nucleotide sequences:
1. The human cancer-related gene comprises one of the following nucleotide sequences:
In the sequence listings, SEQ ID No: 6 is the allelic gene of SEQ ID No: 1, consisting of 2264 bases. Its open reading frame starts from 17 to 1129 base. This sequence contains two tandemly arranged 19 bp DNA segments, the sequence of which is gcttgg agctccagca gct. These 19 bp DNA segments localized in nt 124-nt 161 in SEQ ID No: 6.
The human cancer related LAPTM4B protein possesses the amino acid sequence of 4, and/or 5, and/or 7. Or it consists of the sequence 4, and/or the sequence 5, and/or the sequence 7 after one or several amino acid residues are replaced, deleted, or added. However, the above altered sequence 4, and/or the sequence 5, and/or the sequence 7 still have the same or similar activity to the unchanged sequence 4, and/or the sequence 5, and/or the sequence 7.
Sequence 4 in the sequence listings consists of 317 amino acid residues encoded by the whole sequence of SEQ ID No: 1. Its molecular mass is 35 kDa and the putative isoelectric point is 9.05. Sequence 5 in the sequence listings contains 226 amino acid residues encoded by the segment of bases from 274th to 954th in the SEQ ID No: 1. Its molecular mass is 24 kDa, and the putative isoelectric point is 4.65. The sequence 7 in the sequence listings is a protein containing 370 amino acid residues.
LAPTM4B gene is widely expressed at different levels in sixteen normal tissues. Its transcriptive expression is very high in testis, cardiac muscle, and skeletal muscle, moderate in ovary, kidney, and pancreas, low in liver, spleen, small intestine, large intestine, and thymus, and is very low in lung and peripheral blood cells. In eight fetal tissues, the expression is high in heart, skeletal muscle, and kidney. In fetal livers, it is slightly higher than that in adult livers. However, its expression in some cancerous tissues is significantly upregulated. For instance, the Northern Blot analysis indicates that the transcription level in 87.3% (48/55) human hepatocellular carcinoma tissues is significantly higher than that in fetal livers and normal livers (
LAPTM4B proteins in SEQ ID No: 4, SEQ ID No: 5 and SEQ ID No: 7 have four fragments of membrane-spanning sequences, one N-glycosylation site, a typical lysosome and endosome targeting signals in the cytoplasmic region. They all belong to the protein superfamily of the tetratransmembrane proteins. However, they have various number of phosphorylation sites. The experiment shows that SEQ ID No: 4 LAPTM4B-35 can form a complex in plasma membrane with the integrin α6β1 (Single specific receptor of laminin in the extracellular matrix) and the epidermal growth factor receptor (EGFR) (
The monoclonal and polyclonal antibodies for SEQ ID No: 4 LAPTM4B-35 protein epitopes, such as polyclonal LAPTM4B-EC2232-241-pAb for SEQ ID No: 4 LAPTM4B-35 in the secondary extracellular region, polyclonal antibodies (LAPTM4B-N1-99-pAb and LAPTM4B-N28-37-pAb) for SEQ ID No: 4 LAPTM4B-35 N terminal sequence, and monoclonal antibodies for LAPTM4B are important in studying the effects of LAPTM4B-35 and LAPTM4B-24 in cancer diagnosis and treatment (
SEQ ID No: 8 is the promoter sequence of LAPTM4B gene. To study the regulation of LAPTM4B gene expression, the LAPTM4B gene promoter and the upstream sequence SEQ ID No: 8 are cloned. There are no typical CCAAT (TTGCGCAAT), TATA cassettes in LAPTM4B gene promoter region. But various binding sites of transcription factors exist in the upstream region of LAPTM4B promoter, such as CREBP1/c-Jun, CEBP, PAX2/5/8, GATA, STAT, c-Ets-1, E2F, LYF-1, and c/v-Myb (
In embodiments of this invention, the genome DNA is genotyped in order to determine the relationship between different LAPTM4B genotypes and susceptibility of hepatocellular carcinoma. LAPTM4B has two alleles, LAPTM4B*1 and LAPTM4B*2, i.e., SEQ ID No: 6, is derived by PCR cloning. As shown in
The expression vectors containing sequences described in SEQ ID No: 1, 2, 3, 6, 8, the transfection cell lines containing SEQ ID No: 1, 2, 3, 6, 8 sequences, and the primers amplifying SEQ ID No: 1, 2, 3, 6, 8 are all encompassed by this invention.
57 cases of hepatocellular carcinoma patients, 50 males and 7 females, ranged in age from 35-70. Their average age was 54±6.0. The tissues tested came from surgically excided specimens. The blood samples for the control group were collected from 206 similarly aged people with no symptoms and no cancer according to clinic tests and from 209 new born babies' umbilical veins.
109 esophagus cancer patients, 76 males and 33 females, ranged in age from 30-70. Their average age was 55±5.4. The test tissues came from surgically excided specimens. 116 people with no symptoms and no cancer, as determined by clinical examination, were selected as the control group S. Their blood samples were taken for testing. All the samples were extracted to obtain genomic DNA.
Chi-square (X2) measurement and single factor ANOVA variance were used to treat and analyze the data.
Four types of liver tissues at various proliferation and differentiation status were chosen. They were from normal adult livers (NL, with very little proliferation and high differentiation), fetus livers (FL, at vigorous proliferation and low differentiation), hepatocellular carcinoma (HCC, out of controlled proliferation and abnormal differentiation), and paired non-cancerous livers (PNL, generally is of precancerous stage in an active proliferation status). The Northern Blot analysis was used to detect the transcription of gene in these tissues. RNA samples were extracted from 5 normal adult liver tissues freshly obtained from surgical excision: 5 liver tissues from abortive fetus, 55 HCC tissues, and 55 paired non-cancerous liver tissues. After electrophoretic separation, they were transferred to a nylon film and hybridized by Dig labeled probe. The film was washed at 68° C. and the hybridization signals were developed according to the manual. The results are shown in
By using fluorescence differential display technique, an unknown gene cDNA segment (LC27) was obtained from differential display spectrum in four types of human liver tissues in different proliferation and differentiation status, such as normal adult livers (NL), fetus livers (FL), cancerous livers (HCC), and paired non-cancerous liver (PNL). The LC27 segment (426 bp) was elongated by splicing homogenous sequences according to the EST to the 5′ direction, and followed by RACE (rapid amplification of cDNA ends) and the high temperature RT-PCR techniques. Two full-length cDNA sequences, i.e., SEQ ID No. 2 and 3, were produced, and then confirmed by sequencing and BLAST program analysis.
The sequence of upstream region of the first exon of LAPTM4B gene at 5′ terminal was obtained by biological informatics, and primers F1 and R1 were designed. Using human genomic DNA from HCC as the template, LAPTM4B promoter and the upstream sequence was obtained by PCR using Platinum Pfx DNA polymerase. After Xho I and hind III enzyme cutting, they were inserted into pGL3-Basicvector to form pGL3-PF1, and its sequence was determined (i.e., the test result see portion a of
As shown in
Genome DNA was extracted from blood lymphocytes or cancer tissue samples from surgical excision of hepatocellular carcinoma and esophagus carcinoma according to the standard phenol-chloroform method.
By using the same procedures for the promoter sequence cloning, two primers, F1: 5′ GCGCTCGAGGCTCCAGGTGGAAGAGTGTGC 3′ (SEQ ID No. 11, inducing XhoI enzyme cutting site at 5′ terminal sequence as indicated by underlining), and R1: 5′ GCGAAGCTTGGACTTGGCCATGTGACCCG 3′ (SEQ ID No. 16, inducing XhoI enzyme cutting site at 5′ terminal sequence as indicated by underlining), were designed and synthesized based on LAPTM4B gene sequence SEQ ID No. 3. The promoter sequence and its anterior sequence in the first exon of LAPTM4B were then cloned from human genomic DNA by PCR. The pGL3-PF1 vectors constructed from various human genomic DNA were sequenced to screen the LAPTM4B alleles. The original LAPTM4B sequence was designated as LAPTM4B*1. The other one was designated as LAPTM4B*2, i.e., SEQ ID No. 6 in the sequence listings.
The results reveal that LAPTM4B*1 contains one copied 19 bp DNA sequence and LAPTM4B*2 has two copied 19 bp DNA sequences, which are linked in the non-coded area (nt −33-−15) of the first exon of LAPTM4B*1.
The sequence analyses indicate that LAPTM4B*2 and LAPTM4B*1 possess the same promoter. There is no difference in sequences between LAPTM4B alleles *1 and *2 promoters.
E2 (5′ GCCGACTAGGGGACTGGCGGA 3′, SEQ ID No. 9) and R2 (5′ CGAGAGCTCCGAGCTTCTGCC 3′, SEQ ID No. 10) primers were designed and synthesized. A partial sequence of the first exon of LAPTM4B was amplified by PCR using templates of genomic DNA from normal people, hepatocellular carcinoma, and esophagus carcinoma tissues. PCR conditions were as follows: 96° C. pre-denature for 5 min; 94° C. for 30 s, 68° C. for 30 s, 72° C. for 1 min, 35 cycles; 72° C. for 5 min; then the PCR products were conducted to 2% Agarose gel electrophoresis analysis.
A series of vectors, that contain the upstream sequences with various length of the LAPTM4B promoter, 5′UTR, the 35 bp encoding sequence in exon and the luciferase reporting gene, were constructed, i.e., the LAPTM4B gene promoter and the upstream sequence was cut by Xho I and I Hind III enzyme and connected to pGL3-Basic vector to form pGL3-PF1, and identified by sequencing. Then pGL3-PF1 was used as a template, primers F2, F3, and F4 vs. R1 were used to amplify by PCR, respectively, to construct vectors, pGL3-PF2, pGL3-PF3, and pGL3-PF4 which contain promoter segments with various lengths and luciferase gene. These constructs were identified by sequencing.
The sequences of these primers are as follows:
These vectors were used to transfect BEL-7402 cells and HLE cells separately and the promoter activities were measured. As shown in
The tissue sample was placed on ice and cut into small pieces by scissors. 0.1 gram of wet tissue was selected and placed in a manually operated homogenizer. 1 mL lysis buffer was added in each tube and the mixture was thoroughly homogenized. The lysate was transferred to a fresh tube and centrifuged at 4° C., 12 000 g for 10 min to remove the debris. If cells are used, the cells in a culture dish were digested with 0.25% trypsin buffer, followed by two PBS rinses and centrifuged at 500 g for 3 to min. The cleared supernatant was collected, and the proteins in the supernatant were separated by SDS-PAGE electrophoresis, and then transferred to the NC membrane. The membrane was blocked at 4° C. overnight with 5% non-fat powdered milk in a TBS buffer containing 0.05% Tween 20. Then it was incubated with the rabbit polyclonal antibody, LAPTM4B-EC2 232-241-pAb (1:500 dilution) or mouse Anti-FLAG M2 monoclone antibody (Sigma, 1:750 dilution) at room temperature for 2 hours, and then rinsed with TBS for three times. It was further incubated with a peroxidase-coupled second antibody (IgG), such as goat anti rabbit or goat anti mouse (1:3000 dilution), for 2 hrs, followed by three rinses with a TBS buffer (pH 8.0, containing 0.05% Tween 20). The last wash was with a buffer containing no Tween 20. ECL (Santa Cruz) was used to expose the positive bands (performed as manufacturer's instructions). When two antibodies were sequentially hybridized in one membrane, the ECL exposed membrane was rinsed first with TBS followed by washing with 30 mL TBS (2% SDS and 210 μL β-mercaptoethanol) for 30 min at room temperature. The 30 min TBS rinse removed the previous antibody and its signal in the membrane, which then could be used for the second hybridization.
Using pGEMT-E2E7 plasmid as a template and the PCR method, a full length or partial cDNA, or the reading frame of LAPTM4B gene was amplified by PCR with primers A, or B and E, and the Pfx DNA polymerase. BamHI enzyme cutting site (GGATCC) and ribosome binding site sequences (GCCACC) were introduced in primer A and B at 5′ terminal and EcoRI enzyme cutting site (GAATTC) was incorporated in the primer E. The amplified products AE and BE were digested by restriction enzymes BamHI and EcoRI, purified, and ligated into pcDNA 3.0 vector. They were transformed conventionally to DH5 E. coli and the positive clone was selected, and the constructed plasmid was sequenced for identifying. The constructed plasmids were named as pcDNA3/AE and -BE, respectively. pcDNA3/AE contains a full-length ORF, while pcDNA3/BE contains the ORF starting from the second ATG to TAA. Compared with pcDNA3/AE, pcDNA3/BE-encoded protein is missing 91 amino acids at the N terminal.
Mouse BHK, NIH3T3 cell lines and human hepatocellular carcinoma HLE cell line, in which the expression of LAPTM4B were all at very low level, were transfected by pcDNA3/AE or -BE, and clones that LAPTM4B expression were stable and high were selected. The total viable cell numbers were determined by the acidic phosphatase method and the cell growth curve was plotted. The cell cycle was analyzed by the flow cytometry. The expression levels of cell cycle-regulating protein, including cyclin D1 and Cyclin E, and proto-oncogene products, including c-Myc, c-Fos, and c-Jun (transcription factors for regulating cell proliferation) were measured by the Western Blot analysis. The results show that the cell proliferation was accelerated after being transfected by LAPTM4B-AE expressive plasmid (
Six-week old male mice were randomly selected and divided into three groups: In the first control group, the mice were injected with physiological saline. In the second control group, the mice were inoculated with the pcDNA3 MOCK (no-load plasmid) transfected cells by. In the test group, all the mice were inoculated with pcDNA3/AE (a plasmid containing full-length cDNA) transfected NIH3T3 cells. Each mouse was subcutaneously inoculated with 2×106 cells. There were four to six mice in each group. The mice were sacrificed after 21 days inoculation and dissected. As shown in
The results in Examples 4, 5, and 6 indicate that LAPTM4B may be a novel proto-oncogene.
96 wells culture plates were coated with sera in various dilutions from HCC patients and normal people by known agreement at 4° C. overnight. Each well was washed with 0.5% Tween-20 washing solution, and then 2% BSA was added for blocking at room temperature for 1 hour. Then LAPTM4B-EC2-pAb antibody in various dilutions was added and incubated for 2 hours at room temperature. The goat anti-rabbit antibody labeled by horseradish peroxidase (1:1000 times dilution) was added after PBS washing. After standing at room temperature for 2 hours and one PBS washing, 1 g/mL o-phenyldiamine was added for 10-15 minutes to develop color and H2SO4 was used to stop the reaction. The microtiter for enzyme analysis was used to measure OD. at 490 nm and the antigen level was estimated. The results are shown in
The cell lysate was prepared according to the method in Example 4. The first antibody was added to the supernatant. After 1 hour's shaking at 4° C., 50 μL protein G-Agarose suspension was added and the mixture was shaken at 4° C. for at least three hours or overnight. The immunocomplex precipitate was collected after centrifuging at 12000 g for 20 seconds. The complex was re-suspended by adding 1 mL washing buffer I and shaken at 4° C. for 20 min. The mixture was centrifuged at 12000 g for 20 seconds and the supernatant was removed carefully. This step was repeated once. Then the complex was re-suspended by adding washing buffer II, shaken at 4° C. for 20 min., and centrifuged at 12000 g for 20 seconds. The supernatant was removed carefully. The last two steps were repeated once. The complex was re-suspended by adding washing buffer III, shaken at 4° C. for 20 min, and followed by 12 000 g centrifugation for 20 seconds. The supernatant was removed completely. 50 μL 1×SDS loading buffer was added in the precipitate and the mixture was boiling in 100° C. water bath for 5 min to denature and dissociate the immunocomplex in the sample. After 12000 g centrifugation for 20 second, the supernatant was removed and analyzed in SDS-PAGE apparatus.
BEL-7402 cell was preincubated for 0, 10, 20, and 40 min, respectively, on LN-1 substance in serum free medium. Co-immunoprecipitation was performed with LAPTM4B-EC2-pAb from the cell lysate. The co-immunoprecipitates were respectively adsorbed by Protein G-Sephorose, centrifuged, and analyzed by 10% non-reductive SDS-PAGE. Then the phosphorylations of LAPTM4B, FAK and MAPK were analyzed separately by the Western Blot with p-Tyr mAb.
BEL-7402 cells were preinoculated separately with LAPTM4B-EC2-pAb (15 μg/mL) and anti-Glut2 (15 μL/mL) antibodies at 37° C. under 5% CO2 for 2 hrs, and then seeded on LN-1 substance and incubated for indicating time. Under the same conditions, the anti-Glut2 antibody treated cells and no antibody treated cells were used as control. The cell lysate in each group was analyzed by the Western Blot analysis with p-Tyr mAb. The inhibitory effects of various antibodies on phosphorylation of LAPTM4B were analyzed. The results show that LAPTM4B-35 was phosphorylated peakly when human hepatocellular carcinoma BEL-7402 cells were attached on laminin substrate. The phosphorylation of LAPTM4B-35 reached the highest level in 10 min after cell attachment (
The results from Examples 4-8 suggest that LAPTM4B-35 can be potential targets of drugs for regulating cell proliferation, differentiation, and apoptosis.
LAPTM4B genotypes in genomic DNA from blood of normal individuals and patients with hepatocellular carcinoma were detected by PCR. Two primers were designed and synthesized according to the flanking sequence of 19 bp DNA sequence in LAPTM4B gene sequence 3:
The partial sequence of the first exon was amplified using genomic DNA as a template. PCR conditions were as follows: 96° C. pre-denature for 5 min, 94° C. for 30 sec, 68° C. for 30 sec, 72° C. for 1 min, 35 cycles, 72° C. extension for 5 min. PCR products were analyzed by 2% agarose gel electrophoresis and the results are shown in
In one of the embodiments of the present invention, the occurrence frequency of LAPTM4B genotypes in 209 normal Chinese and 57 patients with hepatocellular carcinoma was analyzed and compared in Table 2. The Hardy-Weinberg equation was used to get the expectancy analysis. The frequency of LAPTM4B allele *1 and *2 from patients with hepatocellular carcinoma differs significantly from that of normal people. Their ratios are 0.5175:0.6746 and 0.4825:0.3254, respectively. The occurrence frequencies of LAPTM4B allele *1 and *2 in a normal population are 0.6746 and 0.3253, while the occurrence frequency of LAPTM4B allele *1 and *2 in patients with hepatocellular carcinoma are 0.5175 and 0.4825. The occurrence frequency of genotype *1/*1 (p=0.029) and *2/*2 (p=0.003) in the group of hepatocellular carcinoma patient shows a significant statistical difference from its control group. In the hepatocellular carcinoma patient group, only 29.8% is of *1/*1, while in the normal control group, 45.93% is of *1/*1. The occurrence frequency of *2/*2 genotype in the hepatocellular carcinoma patient group is 26.32% as compared to 11.01% in the control group, therefore its occurrence frequency is increased significantly (p<0.01). The analysis shows that the risk suffering from HCC of individuals in *2/*2 genotype of is 2.89 times greater than that in other genotype in developing hepatocellular carcinoma. Thus, the LAPTM4B*2/*2 genotype is correlated with the susceptibility of developing hepatocellular carcinoma.
As shown in Table 3, patients with different LAPTM4B genotypes did not show any differences in hepatocellular carcinoma Grade, stage, or HBV infection. 83.3% of the HCC patients have a positive HBV.
a OR: 0.500, 95% CI: 0.267-0.939;
b OR: 2.888, 95% CI: 1.390-6.003 (OR risk suffering HCC, and 95% CI is confidence interval)
To study if the LAPTM4B genotype is related to the susceptibility of developing other cancers, the genomic DNA from blood of 116 normal people and 109 patients with esophagus carcinoma from the same location were analyzed. As shown in Table 4, LAPTM4B genotype of patients with esophagus carcinoma is no significant different from control group of the normal population. LAPTM4B alleles are not related with the susceptibility of developing esophagus cancer.
The relationship between the LAPTM4B-35 protein expression and other cancers was studied by an immunohistochemical method. The fixed specimens from esophagus cancer, breast cancer, lung cancer, stomach cancer, colon cancer, and rectal cancer positive tissues and the negative control noncancerous tissues were obtained from surgical excision and treated according to the following steps:
1. Specimen dewaxing by xylene
2. Katocromy with different concentrations of ethanol, 100%-95%-90%-80%-70%. H2O2 was used to remove endogenous peroxidase
3. Antigen repairing by sodium citrate
4. PBS rinse twice
5. Normal goat serum blocking
6. Keep LAPTM4B-N1-99pAb at 37° C. for 1 hour
7. PBS rinse three times
8. Keep HRP labeled goat anti-rabbit antibody at 37° C. for 1 hour
9. PBS rinse three times
10. Develop color by DAB
11. Nuclear retaining with hematoxylin
12. Ascending dehydration by ethanol at different concentrations (70%-80%-90%-95%-100%
13. Mounting
As shown in
The proteins encoded by LAPTM4B gene in this invention could be possibly used as new markers in early diagnosis of some cancers. By using the widely applied ELISA method in clinical tests, and the prepared related testing reagent kits, the efficiency and the accuracy of the early diagnosis of cancers, especially the primary hepatocellular carcinoma, can be improved.
LAPTM4B gene can be used as target gene in the cancer treatment. Suppressing LAPTM4B-35 expression and promoting LAPTM4B-24 expression could inhibit the growth of hepatocellular carcinoma cells, reverse malignancy phenotype or delay its development. For example, the expression products of LAPTM4B gene, LAPTM4B-35 could be inhibited by the newly developed siRNA interference technology. Furthermore, LAPTM4B-BE-cDNA could be recombinated in the engineered virus expression vector and be used in antitumor gene therapy through an up-regulation of LAPTM4B-24 expression. LAPTM4B-35 protein could also be used as a new target for pharmaceutical treatment. Since LAPTM4B-35 protein can function as an assembling platform for complex of cell signal transduction molecules, and it contains a number of binding sites for signal molecules, there is a great potential to develop various new medicines with LAPTM4B protein as targets. Moreover, this invention has initially demonstrated that LAPTM4B-EC2-pAb antibody can inhibit tumor cell proliferation and block its signal transduction. Based on the discovery in this invention, further studies can be pursued on the possibility of using antibody to inhibit hepatocellular carcinoma and some other cancer development. After a better understanding on the effect, a humanized soluble single chain antibody could be developed for clinical treatment on HCC patients. Peptide vaccines could also be developed. If the vaccines can be successfully made, it will not only help cure hepatocellular carcinoma and some other cancer, but also prevent cancerogenesis in the high risk population. In summary, many new anticancer approaches can be developed based on the embodiments of this invention. As important supplements for treatments of hepatocellular carcinoma and other cancers, this invention will help increase the cure rates of hepatocellular carcinoma and other cancers. This project would generate a significantly great impact on human society.
In specific embodiments, LAPTM4B genotype of genomice DNA is genotyped. The relationship of various genotypes with the susceptibility to hepatocellular carcinoma as well as with other cancers s is investigated. It is discovered that one of the genotypes, LAPTM4B*2/*2, is correlated closely to hepatocellular carcinoma susceptibility. As a result, it provides a new and accurate criterion for screening people who are susceptible to primary hepatocellular carcinoma in the high risk population. It is of important significance to the assessment and prevention of high risk population from developing hepatocellular carcinoma.
| Number | Date | Country | Kind |
|---|---|---|---|
| 02158110.X | Dec 2002 | CN | national |
| 03109786.3 | Apr 2003 | CN | national |
This application is a continuation of U.S. Ser. No. 13/019,297, filed Feb. 1, 2011, incorporated by reference herein in its entirety, which is a divisional of U.S. Ser. No. 10/540,539, filed Oct. 4, 2006, now U.S. Pat. No. 7,910,711, which is a national stage of PCT/CN2003/01109, filed Dec. 24, 2003, which claims the priority benefit of Chinese Application No. 02158110.X, filed Dec. 24, 2002, and Chinese Application No. 03109786.3, filed Apr. 21, 2003.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10540539 | Oct 2006 | US |
| Child | 13019297 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13019297 | Feb 2011 | US |
| Child | 15146307 | US |
