人子宫颈鳞状细胞癌细胞系HCC-0214的建立及其生物学特性

目的 建立人子宫颈鳞状细胞癌细胞系HCC-0214,为子宫颈癌研究提供实验模型。方法 无菌切除人子宫颈癌的手术标本,用组织块贴壁法体外培养,连续传代稳定生长后,绘制细胞生长曲线。采用光镜、电镜观察细胞形态,并进行细胞周期和染色体核形分析。用免疫组化法测定细胞系肿瘤标记物的(ER、PR、Keratine、PCNA)表达情况。结果 组织块贴壁法体外培养获得人子宫颈鳞状细胞癌细胞系HCC-0214(简称H),细胞维持培养16个月,传代131代,生长稳定,群体倍增时间为35．48h,细胞呈上皮镶嵌状贴壁生长,趋向复层生长,无接触抑制。超微结构显示,具有典型的桥粒结构和较多的张力原纤维。染色体数目35—156条,主流范围58—80条(64．8％),结构为人类染色体。细胞的肿瘤标记物(ER、PR、Keratine、PCNA)检测呈高表达,DNA指数为1．931。裸鼠移植瘤组织病理形态学与患者原始肿瘤一致,无血清培养成功。结论 通过组织块贴壁法体外培养建立的人子宫颈鳞状细胞癌细胞系HCC—0214,与原发癌保持相同的生物学特性,体外连续传代16个月以上,细胞形态不变,生长周期恒定,可望作为一个稳定的细胞系。     

Characterization of two human cell lines (TK-10, TK-164) of renal cell cancer

Two previously unreported cell lines of human renal cell carcinoma are presented. TK-10 and TK-164 have each been in culture for over 4 years. The epithelial nature of both cell lines has been documented by light and electron microscopy. The cells in each line contain a Y chromosome, have specific marker chromosomes, and a distinct flow cytometric histogram. Both lines grow in agar, albeit not in athymic mice.     

Establishment of a continuous tumor-cell line (panc-1) from a human carcinoma of the exocrine pancreas.

An epithelioid cell line, started from a human pancreatic carcinoma of ductal cell origin, has been maintained in culture for over 2 years and has been subcultured more than 40 times. The PANC-1 cell line has a doubling time of 52 h and G6PD activity of the slow mobility of B type. Chromosome studies show a modal number of 63 with three distinct marker chromosomes and a small ring chromosome. The malignant nature of the PANC-1 cell line was verified by: (1) the ready growth of PANC-1 cells in soft agar and on top of a fibroblast monolayer; and (2) the formation of a progressively growing anaplastic carcinoma after injection of a nude-athymic mouse with PANC-1 cells.     

Establishment and characterization of continuous cell line MTC-SK derived from a human medullary thyroid carcinoma

Tumor cells of a human medullary thyroid carcinoma were isolated and propagated in tissue culture. Several cell lines with different morphology developed from the primary culture, among others a fibroblast-like growing cell line (MTC-F) and a cell line growing as a suspension of single cells and spherical cell clusters (MTC-SK). The MTC-SK cell line was serially propagated for 90 passages, over 3 years. When examined at different times throughout the in vitro period, MTC-SK exhibited properties characteristic of medullary thyroid carcinomas: the cells maintained their epithelioid morphology; endocrine granules were demonstrated in the cytoplasm by electron microscopy; in situ hybridization confirmed the production of calcitonin- and bombesin-mRNA (gastrin releasing peptide); the cells revealed positive immunoreactivity with antibodies to calcitonin, calcitonin gene-related peptide, and bombesin. The in vitro properties of the MTC-SK cells corresponded to the results obtained from the tissue of origin. Cytogenetic studies of the MTC-F cell line revealed a supernumerary metacentric chromosome (20?). In the MTC-SK cell line the predominant findings were terminal chromosomal rearrangements most frequently concerning chromosome 11p, i.e., the locus of the calcitonin and calcitonin gene-related peptide genes and the H-ras oncogene, and a characteristic instability of the centromeric region of chromosome 16 and somatic pairing of the homologous chromosomes 16.     

具有高转移潜能的人肝癌细胞系的建立及其生物学特性

目的利用裸鼠人肝癌高转移模型（ＬＣＩ－Ｄ２０）的皮下移植瘤组织在体外建立一株具有高转移潜能的人肝癌细胞系（ＭＨＣＣ９７）,并对其一般生物学特性进行观察。方法将分离的瘤细胞制成细胞悬液,用１０％人ＡＢ型血清的高糖ＤＭＥＭ培养液建成该细胞系,采用流式细胞术和染色体Ｇ－显带方法,进行细胞遗传学分析;用ＡＢＣ免疫组化法,观察其肺转移灶中癌细胞甲胎蛋白（ＡＦＰ）表达情况。结果ＭＨＣＣ９７细胞为典型的上皮样细胞,符合一般上皮性恶性肿瘤细胞的病理学特征。该细胞经皮下和肝内接种均可使裸鼠致瘤,并发生肺部转移。肝内接种者,肺转移达１００％（１２／１２）。ＭＨＣＣ９７细胞为异倍体细胞,染色体均为超二倍体,ｉ（１）（ｑ）和ｄｅｒ（４）（ｐｔｅｒ→ｑ３５：：？）等为其标志染色体,未显示有完整Ｙ染色体存在。肺转移灶的癌细胞ＡＦＰ阳性。结论ＭＨＣＣ９７细胞具有与原移植瘤相似的生物学特性。染色体的畸变可能与其发生发展有关     

Cytogenetic analysis of human renal carcinoma cell lines of common origin (NC 65).

Cytogenetic analyses were performed on 3 clonal cell lines derived from a human renal cell carcinoma and its lymph node metastasis, two long-term tissue culture cell lines (NC 65-Sp and NC 65-R) and a serially transplantable tumor line growing on nude mice and brought into culture at the fifth animal passage (NC 65-V). Karyotype were established using banding techniques. Most of the marker chromosomes could be identified and were derived by deletion, inversion, translocation, or isochromosome formation of Chromosomes 1, 3, 4, 5, 8, 9, and 17. These markers were different from HeLa markers. NC 65-Sp had a near diploid chromosome number, NC 65-R a hypotetraploid number, and NC 65-V had a bimodal chromosome number, and NC 65-V had a bimodal chromosome number. Three chromosome markers were shared by the three cell lines; NC 65-R and NC 65-V shared an additional set of four markers. Markers specific to each line were also observed; they demonstrated the independent derivation of the lines and eliminated laboratory cross-contamination. Common markers between the lines confirmed their common tumoral origin.     

Activation of the met oncogene in the human MNNG-HOS cell line involves a chromosomal rearrangement

In this study it is demonstrated that the activated met gene,which was originally detected in the MNNG-HOS chemically transformedhuman cell line, is a chimeric gene formed by the joining togetherof two distinct regions of DNA. Rearrangement of cellular DNAin MNNG-HOS cells was demonstrated by Southern analyses, whichshowed that the MNNG-HOS cell line contained unique met-relatedDNA fragments that were not detected in the parental cell line,HOS. Chromosomal localization using a series of rodent-humanhybrid cell lines showed that the 5' end of the activated metgene is derived from human chromosome 1, in contrast to the3' end of met which has been previously localized to human chromosome7. The chimeric gene is transcribed to produce a 5-kb mRNA thatis encoded both by regions of the gene derived from chromosome1 and by regions of the gene derived from chromosome 7. Karyotypeanalysis of HOS and MNNG-HOS cells has identified several markerchromosomes that involve translocations of chromosomes 1 and7. The possible location of the activated met locus within theserearranged chromosomes is discussed.     

Evolution of human esophageal cancer cell in the process of cell line establishment

An esophageal cancer cell line EC8501 was established by tissue culture technique in vitro. Biologic appraisements demonstrated that this cell line was certainly a malignant one. The authors counted chromosome number of 1,284 cells from 10 to 30 passages and discovered that the modal chromosome number was 46 in 10 and 13 passages, 47 in 14 passage and 65 or so after 25 passage. It was a cell population with subtriploid G-banded chromosome analysis of 73 cells from 7 passages (13, 15, 25-27, 29 and 30) showed that structural chromosome aberrations were manifold, complicated and changeable. Sixteen marker chromosomes were present at appearance rate of 11-97%, 6 of which appeared in every passage. Many derivative chromosomes in the 13-30 passages were derived from marker chromosomes. Thirteen markers were first discovered in the 13 passage and 16 of rearrangement points on the markers were close to 7 oncogenes and 7 cancer breakpoints. Two of thirteen markers (del 1p13 and der 2) were similar or same to markers found in epithelium adjacent to esophageal cancer of two patients. The authors suggested that the two markers may reflect the chromosome changes in early carcinogenesis of esophageal epithelium. According to this research, the authors consider that the tumor cell lines cultured in vitro for many years can not reflect the characteristics of tumor cells in vivo.     

Characterization of a human ovarian adenocarcinoma line, IGROV1, in tissue culture and in nude mice

A cell line, IGROV1, originating from an ovarian carcinoma of a 47-year-old woman was established in tissue culture and in nude mice. Maintained in monolayer cultures, IGROV1 cells exhibited a 20-h doubling time and highly tumorigenic properties. The s.c. injection of 2 X 10(6) cultured cells into nude mice gave rise to fast growing tumors, while the i.p. route induced a peritoneal carcinomatosis with ascites which killed the animals in 2 months. The epithelial morphology of IGROV1 cells was retained during in vitro and in vivo passages, as judged by both the light and the electron microscopes. Two cytogenetic markers characterize IGROV1 cells: a paracentric inversion of chromosome 3, and a translocation between chromosomes 2 and 5. The constitutional karyotype of the patient was normal. These characteristics make the IGROV1 cell line a suitable experimental model for the treatment of human ovarian carcinomas and for biological studies of human solid tumors.     

Alteration of tumorigenicity in undifferentiated thyroid carcinoma cells by introduction of normal chromosome 11

The tumorigenicity of various cell lines has been shown to be suppressed by the introduction of chromosome 11 and other chromosomes via micro-cell-mediated chromosome transfer. In this study, we investigated whether a human undifferentiated thyroid carcinoma cell line, TTA-1, was suppressed by the introduction of normal human chromosome 11 or 10. Chromosome 10 or 11 was transferred from A₉ cells containing a single human chromosome 10 or 11 tagged with pSV₂-neo plasmid DNA into TTA-1 cells, by microcell fusion. The tumorigenicity of the TTA-1 cells and their colony-forming efficiency in soft agar were suppressed by chromosome 11, but not by chromosome 10. These results suggest that normal human chromosome 11 carries a putative tumor suppressor gene that affects the tumor-associated phenotypes of TTA-1 cells. © Wiley-Liss, Inc.     

Characterization of a human ovarian carcinoma cell line, OTN 14, derived from a mucinous cystadenocarcinoma

A human ovarian carcinoma cell line, OTN 14, has been established from malignant ascitic fluid of a patient with a well-differentiated mucinous cystadenocarcinoma of the left ovary. The cell line has been maintained in vitro for 6 months through 23 passages, growing in monolayers as well as in 3-dimensional clusters, with a population doubling time of 28 1/2 hr. The number of chromosomes per cell varied from 67 to 88, with a modal number of 86. Two characteristic marker chromosomes were recognized, consisting of partially deleted chromosome I. With a DNA index of 1.934 the tumour cell line was near tetraploid. The epithelial character of the OTN 14 cells was confirmed by a positive immunofluorescence reaction with monoclonal antibodies (MAbs) against different keratins, and when (immuno)electron microscopy was used, keratin filaments and small junctional complexes were observed. Vimentin was also expressed in these cells, while desmin was not detected. Cultured tumour cells reacted (weakly) positive with MAb OV-TL 3 as a marker for ovarian carcinomas, while reactivity with the anti-ovarian carcinoma MAb OC 125 was limited to a few cells, not permitting the detection of shed CA 125 antigen in the culture supernatant. Cells stained heterogeneously positive for CEA marker BW 431/31, the presence of which was confirmed by detection of CEA shed into the culture medium. The cell line released estradiol at a concentration of 130,000 pmol/L in the culture medium, while no progesterone or dehydroepiandrosterone sulphate were found. Electron microscopical evidence for steroid production was suggested in some cells showing "dense-core" vesicles near the Golgi areas. The OTN 14 tumour cells formed poorly differentiated tumour nodules in nude mice, and metastatic cells were also found in blood capillaries. Cell types with mucinous as well as endocrine characteristics were found.     

Two established in vitro cell lines from human mesenchymal tumours

The development and cultivation of two cell lines from human mesenchymal tumours is described. Lines 2T and 4T differed from normal human fibroblastic cell strains by showing cytological atypia (high nucleocytoplasmic ratio, hyperchromasia and pleo-morphism), an altered “epithelial-like” pavement growth pattern, a deficient inhibition of cell division in crowded cultures and a potential for “indefinite” multiplication in vitro. Both lines were chromosomally abnormal in all samples and differed karyo-typically from any known continuous cell line as well as from each other. Line 2T has retained, at least for 3 months (passage 111–126), a fairly narrow chromosomal mode of 34–38 with two specific marker chromosomes. A high frequency of chromosome breakage was observed in line 2T and extensive chromosome fragmentation, “chromosome pulverization”, occurred. No viruses could, however, be detected in the cultures despite numerous attempts. A control cell strain (2S) was developed from uninvolved skin of one of the tumour patients. Its behaviour followed the general pattern of human diploid cell strains in culture. To our knowledge, the described cell lines are the first derived from human mesenchymal tumours where the availability of a control fibroblast strain will provide us with opportunities to compare autologous normal and neoplastic human mesenchymal cells under in vitro conditions.     

Phenotypic and cytogenetic characterization of a cell line derived from primary prostatic carcinoma

A new cell line, PPC-I, has been established from a specimen obtained from a patient with a poorly differentiated adenocarcinoma of the prostate. This is the first line of its type derived from a primary prostatic tumor site. PPC-I cells have become immortalized in culture, exhibit transformation parameters including relaxed growth factor requirements and anchorage-independent growth, and are highly tumorigenic in nude mice. Cytogenetic studies by G-banding revealed a grossly abnormal karyotype, with a modal chromosome number of 84, multiple marker chromosomes including both homogeneously staining regions and double minutes and clonal loss of chromosomes 3, 5, 10, 15 and Y.     

Multiple chromosomes carrying tumor suppressor activity for a uterine endometrial carcinoma cell line identified by microcell-mediated chromosome transfer

Putative tumor suppressor genes can be mapped to specific chromosomes by the introduction of individual chromosomes derived from normal cells via microcell fusion. We have examined whether a highly malignant human uterine endometrial carcinoma cell line, HHUA, can be suppressed by only one normal chromosome or by multiple chromosomes. A library of mouse A9 clones containing different human chromosomes tagged with the pSV2-neo plasmid DNA were constructed. Transfer by microcell fusion of either chromosome 1, 6, 9, 11, or 19 into the HHUA tumor cell line was performed, and the abilities of the microcell hybrids to form tumors in nude mice were examined. The introduction of a chromosome 19 had no effect on the tumorigenicity of the cells, whereas microcell-hybrid clones with an introduced chromosome 1, 6 or 9 were completely suppressed for tumorigenicity. A decrease in tumor-take incidence in some but not all clones was observed following the introduction of a chromosome 11. The nontumorigenic microcell hybrids with an introduced chromosome 1 differed from the nontumorigenic microcell hybrids with an introduced chromosome 6, 9, or 11. A large percentage of hybrids with chromosome 1 senesced and/or showed alterations in cellular morphology and transformed growth properties in vitro. No growth or morphology alterations were observed following transfer of the other chromosomes. These results may indicate that more than one chromosome carries a tumor suppressor gene(s) for this human uterine endometrial carcinoma cell line and support the hypothesis that multiple tumor suppressor genes control the tumorigenic phenotype in the multistep process of neoplastic development.     

G-banding and high resolution chromosome study of a human lung adenocarcinoma cell line LGC-7910

G-banding and high resolution chromosome banding techniques were used in studying a human lung adenocarcinoma cell line LGC-7910. One hundred cells were counted at Passage 150. Ten, twelve and ten cells were analyzed at Passages 150, 173 and 183, respectively. The model number of chromosome in this cell line was 67-69. A number of marker chromosomes related to complex chromosomal rearrangement was observed. The analysis of chromosomes at the different passages showed a stability during subculture of the cell line. It was found that the cell line had partial deletion of the short arm on chromosome 7 (with breakpoint at 7p15.3-p13), rearrangement of the arm on chromosome 1 (with breakpoint at 1p13.1) and increase in chromosome 7 number. The results suggested that such chromosome alteration be related to the expression of activated oncogenes during carcinogenesis.     

Targeting the Mitotic Checkpoint to Kill Tumor Cells

One of the most common hallmarks of cancer cells is aneuploidy or an abnormal number of chromosomes. This abnormal chromosome content is a consequence of chromosome missegregation during mitosis, a defect that is seen more frequently in tumor cell divisions as in normal cell divisions. In fact, a large fraction of human tumors display a chromosome instable phenotype, meaning that they very frequently missegregate chromosomes. This can cause variegated aneuploidy within the tumor tissue. It has been argued that this hallmark of cancer could be exploited in anti-cancer therapies. Here we test this hypothesis by inactivation of the mitotic checkpoint through RNAi-mediated depletion of an essential checkpoint component, Mps1. The mitotic checkpoint delays segregation of chromosomes during mitosis until all chromosomes are properly attached to the mitotic spindle. Its inactivation will therefore lead to increased segregation errors. Indeed, we show that this can lead to increased cell death in tumor cells. We demonstrate that increased cell death is associated with a dramatic increase in segregation errors. This suggests that inhibition of the mitotic checkpoint might represent a useful anti-cancer strategy.     

Establishment of a cell line (HCC-M) from a human hepatocellular carcinoma

A continuous human cell line was established from a hepatocellular carcinoma of an HBsAg-positive Japanese male. The cell line, designated HCC-M, grows as an adhering monolayer with a doubling time of 24 h in medium RPMI-1640 supplemented with 10% FCS and grows with 30% clonal efficiency in soft agar. The cells have been shown by light and electron microscopy to be of epithelial type. When they were transplanted subcutaneously into the back of athymic nude mice (BALB/c, nu/nu), tumors developed at the sites of inoculation, which were shown to be hepatocellular carcinoma, similar in morphology to the original tumor from which they were derived. HCC-M had a chromosome mode of 63 with five identifiable marker chromosomes. HCC-M produced albumin at the 10th passage but this property was lost by the 30th passage. This cell line has not secreted alpha-fetoprotein. Hepatitis B viral particles or HBsAg have not been demonstrated in the cells from the primary culture nor in several subsequent subcultures tested.     

Growth and transformation suppressor genes for BHK Syrian hamster cells on human chromosomes 1 and 11.

To map putative tumor suppressor genes for the near-diploid baby hamster kidney fibrosarcoma cell line BHK, we transferred five different normal human chromosomes (1, 3, 7, 11, and 12) into these tumor cells by microcell-mediated chromosome transfer. Transfer of human chromosome 1 into BHK cells resulted in suppression of cell growth both on plastic and in soft agar, indicating that chromosome 1 has a generalized effect on cell growth and thereby suppresses anchorage-independent growth. Selection against cells with an intact chromosome 1 was observed. In contrast, the introduction of chromosome 11 into BHK cells resulted in suppression of anchorage independence but not growth on plastic. Most chromosome-11 growth-suppressed BHK hybrids retained intact copies of human chromosome 11. Tumorigenic derivatives of chromosome 11 hybrids had lost this chromosome. Transfer of human chromosome 3, 7, or 12 into BHK cells did not correlate with growth suppression of BHK cells on plastic or in soft agar. Thus, we conclude that genes that suppress BHK-cell growth in general or in agar reside on human chromosomes 1 and 11, respectively.