Aging is a decelerating unidirectional process of life. Shortening of telomeric DNA, the (TTAGGG)<sub>n</sub> hexanucleotide repeats, which form the caps at the chromosome ends, is implicated to determine ...Aging is a decelerating unidirectional process of life. Shortening of telomeric DNA, the (TTAGGG)<sub>n</sub> hexanucleotide repeats, which form the caps at the chromosome ends, is implicated to determine the aging process, and more importantly the healthy lifespan itself. Telomerase, a ribonucleoprotein having reverse transcriptase activity, arrests telomere loss through addition of the TTAGGG repeats de novo, to the ends of the chromosome. The telomere/telomerase maintenance is an inevitable necessity to delay aging and for a healthy lifespan. Here, we report the potential of full-spectrum, high concentration Ashwagandha (Withania somnifera), an Ayurvedic medicinal herb, root extract to increase telomerase activity. HeLa cells, when treated with various concentrations of Ashwagandha root extract, showed an increase in telomerase activity measured with the established Telomerase Rapid Amplification Protocol (TRAP) assay. Ashwagandha root extract increased telomerase activity with highest enhancement of ~45% at 10 - 50 μg concentration. Thus, Ashwagandha root extract has the anti-aging inducing potential.展开更多
This work was supposed by CMB (No. 96—635) This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997). Although cervical carcinoma cells may express the hu...This work was supposed by CMB (No. 96—635) This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997). Although cervical carcinoma cells may express the human papillomavirus protein E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. Recent studies suggest that expression of CD 80 (B7 1) on tumor cells is effective to induce antitumor immune responses. 1,2 In our study, CD 80 gene was transfected into human Hela cell line with a CD 80 expression plasmid (B7 1 +pcDNA 3) by electroporation, then the immunogenecity of the modified Hela cell was tested in TLMC (tumor lymphocyte mixed culture) system. Thymidine lymphocyte proliferation assays showed that the response of human peripheral blood lymphocytes (PBLS) to CD 80 positive Hela cells demonstrated a substantial increase in cell proliferation compared to the response to control cells. Cocultivation of allogeneic PBLs with CD 80 positive tumor cells for three days can induce an increased secretion of IL 2. Our results demonstrate an immunostimulatory effect of CD 80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.展开更多
The chromosomal number variations & structural aberrations of the MDCK cell line, primary feline or canine kidney cell(FKC or CKC) and Hela cell line were investigated and their karyotypes of conventional chromoso...The chromosomal number variations & structural aberrations of the MDCK cell line, primary feline or canine kidney cell(FKC or CKC) and Hela cell line were investigated and their karyotypes of conventional chromosome bands were analyzed. The carcinogenesis or tumorigenicity testing of these cell lines in about 232 nude mice and for colony formation in soft agarose and for haemagglutination under different concentration of plant lectins of these cells were carried out. Under the prerequisite that the incidence of cancer or tumor in negative-control nude mice inoculated subcutaneously with primary feline or canine kidney cell cultures purified in vitro at passage 3 was 0 (0/22) and 0 (0/10), respectively. The incidence of the progressively-growing malignant tumor(MT) in positive-control nude mice inoculated subcutaneously with Hela cell cultures of KB, X, or NM20/X strain was 10/10, 25/25 and 5/51, respectively. The results showed that the incidence of tumor in nude mice with tetraploid YA strain of MDCK cell during 20 - 45 passages, with hy-podiploid JB strain of MDCK cell on passage 25, with di-and hypoploid JC strain of MDCK cell during 2-15 passages or with hypoploid M strain of MDCK cell during 9 - 27 passages was 28/58, 1/5, 4/18 and 0/31, respectively. The chromosomal analysis results showed that the ratio of difference in the rate of modal chromosome number between high(mcs+ n) and lowest (mcs) passages was not more than 5% - 15% and the structure aberrations was generally 0 - 3% . These results proved that the genetic characteristics of chromosomal number of cell lines determines their tumorigenicity, but it is species-specific. MDCK line has tumorigenicity no matter what its chromosome karyotype is, at least it has very low tumorigenicity even when its modal chromosome number is hypoploid. The repeatedly frozen, thawed and split controls of tumorigenicity-positive cell lines(X strain of Hela, M strain of BHK-21, JA strain of Vero, YA strain of MDCK) have much lower tumorigenicity or are even non-carcinogenesis, and the repeatedly frozen, thawed and split controls of very low tumorigenicity cell lines (M or JC strain of MDCK) are certainly non-carcinogenic and never have increased tumorigenicity. It is thus evident that MDCK cell of M, JB or JC strain can be approved as substrate for the preparation of attenuated viral vaccines, but MDCK cell of YA strain can not be approved as substrate for the preparation of attenuated viral vaccines. In summary, all strains of MDCK cell line have tunorigenicity, at least have low tumor igencity , never have non-cancinogenic MDCK, but very low tumorigenicity MDCK cell strains can certainly be used for the approval production of canine viral vaccines if the DNA content in viral cell cultures was remarkably decreased through conventional means in manufacturing process. Therefore, the master cell stock and working cell bank of MDCK line used for vaccine manufacture were established in China, which are free of infectious agents, and described with respect to cytogenetic characteristics and tumorigenicity.Tests showed that there were correlations among cell line chromosome number variations, anchorage independence in soft a-garose, haemagglutination under plant lectins, and tumor-forming ability in nude mice, thus all the in vitro tests are economic, simple and reliable means for monitoring the tumor-forming ability of MDCK line in nude mice.展开更多
Objective To explore the effects and mechanisms of cytoplasmic M-CSF on the proliferation,migration and invasion of HeLa cells.Methods Both pCMV/cyto/myc vector and pCMV/cyto/myc-M-CSF vector was transfected into HeLa...Objective To explore the effects and mechanisms of cytoplasmic M-CSF on the proliferation,migration and invasion of HeLa cells.Methods Both pCMV/cyto/myc vector and pCMV/cyto/myc-M-CSF vector was transfected into HeLa-cell by transfectaimine.After screening by G418,the positive clones were amplified and confirmed by RT-PCR,Western blot and immunocytochemistry.The effect of cytoplasmic M-CSF on the proliferation of HeLa cells were analyzed by cell conuting and antisense oligonucleotides.The migration and invasion of cell was measured by in vitro Transwell assay and Matrigel-coated polycarbonate filters.The expression of cyclinE,cyclinD1/2/3,CDK2/4/6,Rac1,and matrix metalloproteinase 2 and 9(MMP2/9)were assayed by semiquantitative RT-PCR.And expression of both α-tubulin and cdc42 were displayed by immunofluorescence.The activity of MMP2 was detected by gelatin zymography.Results Results A cell line(referred as to HeLa-M cell)that highly expresses cytoplasmic M-CSF was successfully established in the test.Our result indicated that HeLa-M cell had a larger volume,faster growth rate and shorter doubling time than either pCMV/cyto/myc transfected HeLa cells(referred as to HeLa-C cell)or untransfected HeLa cells(referred as to HeLa cell).M-CSF-specific antisense oligonucleoside significantly inhibited HeLa-M cell proliferation and had little effect on either HeLa-C cell or HeLa-C cell growth.Cytoplasmic M-CSF up-regulated both the expression of cyclinE,cyclinD1 and cyclinD3,CDK2,CDK 4 and CDK6,a Rho GTPase ralative protein(Rac1),cdc42 and MMP2,but had little effect on expression of MMP9 and cyclin D2.Furthermore,cytoplasmic M-CSF induced the rearrangement of the α-tubulin in HeLa cells and significantly promoted the migration and invasion of HeLa cells in vitro.Conclusions Cytoplasmic M-CSFs up-regulate the expression of cyclinE,cyclinD1 and cyclinD3,CDK2,CDK 4 and CDK6 and induces the proliferation of HeLa cells.Cytoplasmic M-CSFs up-regulate the expression of Rac1 and cdc42 and cause the rearrangement of the α-tubulin in HeLa cells.Furthermore Cytoplasmic M-CSFs increase both the expression and activity of MMP2 and promote the migration and invasion of HeLa cell in vitro.But cytoplasmic M-CSFs have little effect on expression of cyclin D2 and MMP9.展开更多
The effects of growth factors and calcium concentrations present in different culture media on induction of terminal differentiation were investigated for four different epidermoid carcinoma cell lines, Hela, KB, A431...The effects of growth factors and calcium concentrations present in different culture media on induction of terminal differentiation were investigated for four different epidermoid carcinoma cell lines, Hela, KB, A431, and SCC-25, and their responses determined relative to those elicited by normal human keratinocytes subjected to these culture conditions. Differentiation status was determined cyto-chemically by a validated keratin protein staining method, and by autoradiographic analyses. Growth and differentiation promoting factors that influenced the direction of integrated control of growth and differentiation in normal human keratinocytes were found to be effective for some cell lines but not others. The factors examined were 1) high density arrest in serum-free and serum-containing media, 2) media shifts from high density culture in serum-containing media to low density growth factor-depleted or supplemented serum-free medium, and 3) the concentration of calcium in the media. The extent and degree of differentiation achieved varied among different cell lines depend on the presence or absence of serum, EGF and insulin protein growth factors. Certain growth media appear to sponsor keratin protein, cyto-chemically-detected differentiation, and evidence of quantal mitotic division in low density HeLa cell and SCC25 cell cultures. Epidermoid carcinoma cell lines retain limited capacity to commit to early stages of cell differentiation.展开更多
目的:探讨叉头框转录因子M1(FoxM1)对人宫颈癌顺铂耐药株Hela/DDP增殖、侵袭、凋亡的影响及其机制。方法:将Hela/DDP细胞分为空白组(常规培养,不予任何处理)、正常对照(NC)组(转染空白干扰质粒)、低表达组和过表达组。低表达组转染siRNA...目的:探讨叉头框转录因子M1(FoxM1)对人宫颈癌顺铂耐药株Hela/DDP增殖、侵袭、凋亡的影响及其机制。方法:将Hela/DDP细胞分为空白组(常规培养,不予任何处理)、正常对照(NC)组(转染空白干扰质粒)、低表达组和过表达组。低表达组转染siRNA-FoxM1下调Hela/DDP细胞中FoxM1表达,过表达组转染pcDNA3.1-FoxM1上调Hela/DDP细胞中FoxM1表达。采用RT-qPCR法检测FoxM1 m RNA表达水平,CCK-8检测细胞活力,流式细胞仪检测细胞凋亡,Transwell小室检测细胞侵袭能力。结果:FoxM1 mRNA在Hela细胞中的表达水平低于Hela/DDP细胞(P<0.05)。空白组与NC组中FoxM1 mRNA、增殖率、凋亡率、侵袭细胞数及HSP70、S100A9蛋白表达水平比较,差异均无统计学意义(P>0.05)。与空白组比较,低表达组细胞活力和侵袭能力及FoxM1 mRNA、HSP70蛋白表达水平降低,凋亡率和S100A9蛋白表达水平升高(P<0.05),与空白组比较,过表达组细胞活力和侵袭能力及FoxM1 m RNA、HSP70蛋白表达水平升高,凋亡率和S100A9蛋白表达水平降低(P<0.05)。与低表达组比较,过表达组细胞活力和侵袭能力及FoxM1 mRNA、HSP70蛋白表达水平升高,凋亡率和S100A9蛋白表达水平降低(P<0.05)。结论:下调FoxM1表达可抑制HSP70表达,促进S100A9表达,调控宫颈癌细胞恶性行为,从而提高宫颈癌顺铂耐药细胞株对顺铂化疗的敏感性。展开更多
文摘Aging is a decelerating unidirectional process of life. Shortening of telomeric DNA, the (TTAGGG)<sub>n</sub> hexanucleotide repeats, which form the caps at the chromosome ends, is implicated to determine the aging process, and more importantly the healthy lifespan itself. Telomerase, a ribonucleoprotein having reverse transcriptase activity, arrests telomere loss through addition of the TTAGGG repeats de novo, to the ends of the chromosome. The telomere/telomerase maintenance is an inevitable necessity to delay aging and for a healthy lifespan. Here, we report the potential of full-spectrum, high concentration Ashwagandha (Withania somnifera), an Ayurvedic medicinal herb, root extract to increase telomerase activity. HeLa cells, when treated with various concentrations of Ashwagandha root extract, showed an increase in telomerase activity measured with the established Telomerase Rapid Amplification Protocol (TRAP) assay. Ashwagandha root extract increased telomerase activity with highest enhancement of ~45% at 10 - 50 μg concentration. Thus, Ashwagandha root extract has the anti-aging inducing potential.
文摘This work was supposed by CMB (No. 96—635) This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997). Although cervical carcinoma cells may express the human papillomavirus protein E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. Recent studies suggest that expression of CD 80 (B7 1) on tumor cells is effective to induce antitumor immune responses. 1,2 In our study, CD 80 gene was transfected into human Hela cell line with a CD 80 expression plasmid (B7 1 +pcDNA 3) by electroporation, then the immunogenecity of the modified Hela cell was tested in TLMC (tumor lymphocyte mixed culture) system. Thymidine lymphocyte proliferation assays showed that the response of human peripheral blood lymphocytes (PBLS) to CD 80 positive Hela cells demonstrated a substantial increase in cell proliferation compared to the response to control cells. Cocultivation of allogeneic PBLs with CD 80 positive tumor cells for three days can induce an increased secretion of IL 2. Our results demonstrate an immunostimulatory effect of CD 80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.
文摘The chromosomal number variations & structural aberrations of the MDCK cell line, primary feline or canine kidney cell(FKC or CKC) and Hela cell line were investigated and their karyotypes of conventional chromosome bands were analyzed. The carcinogenesis or tumorigenicity testing of these cell lines in about 232 nude mice and for colony formation in soft agarose and for haemagglutination under different concentration of plant lectins of these cells were carried out. Under the prerequisite that the incidence of cancer or tumor in negative-control nude mice inoculated subcutaneously with primary feline or canine kidney cell cultures purified in vitro at passage 3 was 0 (0/22) and 0 (0/10), respectively. The incidence of the progressively-growing malignant tumor(MT) in positive-control nude mice inoculated subcutaneously with Hela cell cultures of KB, X, or NM20/X strain was 10/10, 25/25 and 5/51, respectively. The results showed that the incidence of tumor in nude mice with tetraploid YA strain of MDCK cell during 20 - 45 passages, with hy-podiploid JB strain of MDCK cell on passage 25, with di-and hypoploid JC strain of MDCK cell during 2-15 passages or with hypoploid M strain of MDCK cell during 9 - 27 passages was 28/58, 1/5, 4/18 and 0/31, respectively. The chromosomal analysis results showed that the ratio of difference in the rate of modal chromosome number between high(mcs+ n) and lowest (mcs) passages was not more than 5% - 15% and the structure aberrations was generally 0 - 3% . These results proved that the genetic characteristics of chromosomal number of cell lines determines their tumorigenicity, but it is species-specific. MDCK line has tumorigenicity no matter what its chromosome karyotype is, at least it has very low tumorigenicity even when its modal chromosome number is hypoploid. The repeatedly frozen, thawed and split controls of tumorigenicity-positive cell lines(X strain of Hela, M strain of BHK-21, JA strain of Vero, YA strain of MDCK) have much lower tumorigenicity or are even non-carcinogenesis, and the repeatedly frozen, thawed and split controls of very low tumorigenicity cell lines (M or JC strain of MDCK) are certainly non-carcinogenic and never have increased tumorigenicity. It is thus evident that MDCK cell of M, JB or JC strain can be approved as substrate for the preparation of attenuated viral vaccines, but MDCK cell of YA strain can not be approved as substrate for the preparation of attenuated viral vaccines. In summary, all strains of MDCK cell line have tunorigenicity, at least have low tumor igencity , never have non-cancinogenic MDCK, but very low tumorigenicity MDCK cell strains can certainly be used for the approval production of canine viral vaccines if the DNA content in viral cell cultures was remarkably decreased through conventional means in manufacturing process. Therefore, the master cell stock and working cell bank of MDCK line used for vaccine manufacture were established in China, which are free of infectious agents, and described with respect to cytogenetic characteristics and tumorigenicity.Tests showed that there were correlations among cell line chromosome number variations, anchorage independence in soft a-garose, haemagglutination under plant lectins, and tumor-forming ability in nude mice, thus all the in vitro tests are economic, simple and reliable means for monitoring the tumor-forming ability of MDCK line in nude mice.
文摘Objective To explore the effects and mechanisms of cytoplasmic M-CSF on the proliferation,migration and invasion of HeLa cells.Methods Both pCMV/cyto/myc vector and pCMV/cyto/myc-M-CSF vector was transfected into HeLa-cell by transfectaimine.After screening by G418,the positive clones were amplified and confirmed by RT-PCR,Western blot and immunocytochemistry.The effect of cytoplasmic M-CSF on the proliferation of HeLa cells were analyzed by cell conuting and antisense oligonucleotides.The migration and invasion of cell was measured by in vitro Transwell assay and Matrigel-coated polycarbonate filters.The expression of cyclinE,cyclinD1/2/3,CDK2/4/6,Rac1,and matrix metalloproteinase 2 and 9(MMP2/9)were assayed by semiquantitative RT-PCR.And expression of both α-tubulin and cdc42 were displayed by immunofluorescence.The activity of MMP2 was detected by gelatin zymography.Results Results A cell line(referred as to HeLa-M cell)that highly expresses cytoplasmic M-CSF was successfully established in the test.Our result indicated that HeLa-M cell had a larger volume,faster growth rate and shorter doubling time than either pCMV/cyto/myc transfected HeLa cells(referred as to HeLa-C cell)or untransfected HeLa cells(referred as to HeLa cell).M-CSF-specific antisense oligonucleoside significantly inhibited HeLa-M cell proliferation and had little effect on either HeLa-C cell or HeLa-C cell growth.Cytoplasmic M-CSF up-regulated both the expression of cyclinE,cyclinD1 and cyclinD3,CDK2,CDK 4 and CDK6,a Rho GTPase ralative protein(Rac1),cdc42 and MMP2,but had little effect on expression of MMP9 and cyclin D2.Furthermore,cytoplasmic M-CSF induced the rearrangement of the α-tubulin in HeLa cells and significantly promoted the migration and invasion of HeLa cells in vitro.Conclusions Cytoplasmic M-CSFs up-regulate the expression of cyclinE,cyclinD1 and cyclinD3,CDK2,CDK 4 and CDK6 and induces the proliferation of HeLa cells.Cytoplasmic M-CSFs up-regulate the expression of Rac1 and cdc42 and cause the rearrangement of the α-tubulin in HeLa cells.Furthermore Cytoplasmic M-CSFs increase both the expression and activity of MMP2 and promote the migration and invasion of HeLa cell in vitro.But cytoplasmic M-CSFs have little effect on expression of cyclin D2 and MMP9.
文摘The effects of growth factors and calcium concentrations present in different culture media on induction of terminal differentiation were investigated for four different epidermoid carcinoma cell lines, Hela, KB, A431, and SCC-25, and their responses determined relative to those elicited by normal human keratinocytes subjected to these culture conditions. Differentiation status was determined cyto-chemically by a validated keratin protein staining method, and by autoradiographic analyses. Growth and differentiation promoting factors that influenced the direction of integrated control of growth and differentiation in normal human keratinocytes were found to be effective for some cell lines but not others. The factors examined were 1) high density arrest in serum-free and serum-containing media, 2) media shifts from high density culture in serum-containing media to low density growth factor-depleted or supplemented serum-free medium, and 3) the concentration of calcium in the media. The extent and degree of differentiation achieved varied among different cell lines depend on the presence or absence of serum, EGF and insulin protein growth factors. Certain growth media appear to sponsor keratin protein, cyto-chemically-detected differentiation, and evidence of quantal mitotic division in low density HeLa cell and SCC25 cell cultures. Epidermoid carcinoma cell lines retain limited capacity to commit to early stages of cell differentiation.
文摘目的:探讨叉头框转录因子M1(FoxM1)对人宫颈癌顺铂耐药株Hela/DDP增殖、侵袭、凋亡的影响及其机制。方法:将Hela/DDP细胞分为空白组(常规培养,不予任何处理)、正常对照(NC)组(转染空白干扰质粒)、低表达组和过表达组。低表达组转染siRNA-FoxM1下调Hela/DDP细胞中FoxM1表达,过表达组转染pcDNA3.1-FoxM1上调Hela/DDP细胞中FoxM1表达。采用RT-qPCR法检测FoxM1 m RNA表达水平,CCK-8检测细胞活力,流式细胞仪检测细胞凋亡,Transwell小室检测细胞侵袭能力。结果:FoxM1 mRNA在Hela细胞中的表达水平低于Hela/DDP细胞(P<0.05)。空白组与NC组中FoxM1 mRNA、增殖率、凋亡率、侵袭细胞数及HSP70、S100A9蛋白表达水平比较,差异均无统计学意义(P>0.05)。与空白组比较,低表达组细胞活力和侵袭能力及FoxM1 mRNA、HSP70蛋白表达水平降低,凋亡率和S100A9蛋白表达水平升高(P<0.05),与空白组比较,过表达组细胞活力和侵袭能力及FoxM1 m RNA、HSP70蛋白表达水平升高,凋亡率和S100A9蛋白表达水平降低(P<0.05)。与低表达组比较,过表达组细胞活力和侵袭能力及FoxM1 mRNA、HSP70蛋白表达水平升高,凋亡率和S100A9蛋白表达水平降低(P<0.05)。结论:下调FoxM1表达可抑制HSP70表达,促进S100A9表达,调控宫颈癌细胞恶性行为,从而提高宫颈癌顺铂耐药细胞株对顺铂化疗的敏感性。
