Exceptional lethality for nude mice of cells derived from a primary human melanoma

BRO human melanoma cells, obtained from a biopsy of a highly aggressive and malignant primary tumor, were grown as xenografts in nude mice and in cell culture. These cells were exceptionally tumorigenic and malignant for nude mice. NIH-II nude mice survived 11.0 +/- 0.4 (S.E.) and 14.1 +/- 0.4 days after i.p. inoculation of 10(7) or 10(6) BRO cells, respectively, and lethal tumors developed in all mice inoculated i.p. with only 10(3) cells. The doubling time (2.3 days) of the volume of tumors formed after s.c. inoculation was comparable to the doubling time of these cells in culture. After i.p. or s.c. inoculation, BRO cells metastasized to the diaphragm and lungs, causing respiratory failure in most of the host mice. The original tumor and the cell line derived from it had undifferentiated structures with prominent nuclei and very large nucleoli. Karyotype abnormalities included a gigantic A group chromosome, a large D group chromosome, and an unusual double centromere chromosome not found typically in human melanoma cells. Due to the short and reproducible survival times of nude mice inoculated i.p. with BRO cells, this model system may be useful for rapidly determining the effects of experimental treatment on the survival of hosts bearing human tumor cells.     

Characterization of six cell lines established from human pancreatic adenocarcinomas

BACKGROUND: Six human pancreatic carcinoma cell lines, designated as KMP-1 to KMP-6, were established and maintained in vitro for > 3 years. All were derived from pancreatic ductal adenocarcinomas. The six cell lines originated from either primary pancreatic tumors, metastatic liver tumors, or metastases to lymph nodes. METHODS: Each cell line was characterized by its morphology, doubling time, colony forming efficiency (CFE) on plastic dishes, tumorigenicity in nude mice, chromosomal analysis, and the amount of tumor markers secreted into the culture medium. Furthermore, mutations in the K-ras, p53, and p16/INK4a genes were analyzed. RESULTS: All cell lines grew as an adhering monolayer and were cultured in medium supplemented with 2% fetal bovine serum. The doubling time ranged from 16-70 hours, and the CFE ranged from 0.1-11%. Subcutaneous transplantation of these carcinoma cells into nude mice resulted in the formation of tumors. Chromosomal analysis showed that the modal numbers ranged from 43-124, and each karyotype was unique. Each cell line secreted detectable amounts of squamous cell carcinoma antigen, carcinoembryonic antigen, carbohydrate antigen 19-9, Dupan-II, and cytokeratin 19 fragment, respectively. Genetic alterations of the K-ras, p53, and p16 genes were detected in six, three, and five, respectively, of the six cell lines. CONCLUSIONS: The authors believe that these newly established pancreatic carcinoma cell lines will contribute to wide ranging studies regarding pancreatic carcinoma progression.     

Establishment and characterization of a human pancreatic cancer cell line (SUIT-2) producing carcinoembryonic antigen and carbohydrate antigen 19-9

A new tumor cell line (SUIT-2) derived from a metastatic liver tumor of human pancreatic carcinoma has been established in tissue culture and in nude mice, and maintained for over five years. In tissue culture, the cells grew in a monolayered sheet with a population doubling time of about 38.2 hr, and floated or piled up to form small buds above the monolayered surface in relatively confluent cultures. Chromosome counts ranged from 34 to 176 with a modal number of 45. Subcutaneous injection of cultured cells into nude mice resulted in tumor formation, histopathologically closely resembling the original neoplasm which had been classified as moderately differentiated tubular adenocarcinoma. Electron microscopic observation of the neoplastic cells revealed a characteristic pancreatic ductal epithelium. SUIT-2 cell line produces and releases at least two tumor markers, carcinoembryonic antigen and carbohydrate antigen 19-9, propagates even in serum-free medium, and metastasizes to the regional lymph nodes in nude mice xenografts.     

ESTABLISHMENT OF A HUMAN PANCREATIC ADENOCARCINOMA CELL LINE （JF305） WITH p53 EXPRESSION

李昕，王宏，姜奕，王晓华，贾兰玲，张宝庚ＥＳＴＡＢＬＩＳＨＭＥＮＴＯＦＡＨＵＭＡＮＰＡＮＣＲＥＡＴＩＣＡＤＥＮＯＣＡＲＣＩＮＯＭＡＣＥＬＬＬＩＮＥ（ＪＦ３０５）ＷＩＴＨｐ５３ＥＸＰＲＥＳＳＩＯＮ￥ＬｉＸｉｎ；ＷａｎｇＨｏｎｇ；ＪｉａｎｇＹｉ；Ｗａｎｇ...     

Development and characterization of a human sarcoma cell line, MES-SA, sensitive to multiple drugs

A cell line designated MES-SA has been developed from a uterine sarcoma. Cells from the surgical tumor specimen were grown in a soft-agar clonogenic assay, with a relatively high plating efficiency of 0.5% and sensitivity to multiple drugs. Histologically, the surgical specimen and tumors developing after MES-SA inoculation into nude mice were identical, consisting of sheets of anaplastic sarcoma cells amid scant hyalinized stroma. The nonepithelial origin of this line was supported by ultrastructural analysis and negative mucin staining. Growth in monolayer was established by seeding colonies from soft agar into liquid media and has been maintained for over 21 months (greater than 100 passages), with a population-doubling time for the cell line of 22 hr. The MES-SA line readily forms colonies in soft agar with plating efficiencies ranging from 10 to 20%. Tumor cell inoculation s.c. into nude mice produces tumors within 2 to 3 weeks and subsequent tumor volume-doubling times of 7 to 10 days. MES-SA has a modal chromosome number of 45. Karyotypic abnormalities include: monosomic forms of chromosomes 5, 6, and 7; a 5q, 6p translocation; and one marker chromosome. In vitro sensitivities to doxorubicin, dactinomycin, mitomycin C, and bleomycin have been demonstrated by clonogenic assay. These drug sensitivities remain stable over long periods of monolayer growth and after passage in nude mice.     

Establishment and characterization of a carcinoembryonic antigen (CEA)-producing cell line from a human carcinoma of the exocrine pancreas

A human carcinoembryonic antigen (CEA)-producing cell line, T3M-4, has been established from explant cultures of a primary human pancreatic exocrine adenocarcinoma transplanted into nude mice. The tumor had metastasized in the patient. The tumor obtained from metastatic lymph nodes was the initial source for implantation in athymic nude mice. In the primary culture, host fibroblasts were eliminated by the use of the antiserum raised against nude mouse cells. T3M-4 cells have been continuously propagated in vitro during the past 26 months. The cells grew in a monolayered sheet with about 31 hours of population doubling time. The cells exhibited epithelial morphologic features resembling the structure of the original tumor, and they showed tumor takes when inoculated into athymic nude mice. Xenografts established from the cell line have retained a similar histology to the original tumor on serial transplantation. Chromosomal analysis revealed the cell line to be a human aneuploid one with a hyperdiploid mode. T3M-4 cells possess the characteristic function of CEA secretion in vitro in culture and in vivo in nude mice bearing the tumors produced by inoculation with the cultured cells. In view of these characteristics, T3M-4 cell line represents a new human pancreatic exocrine adenocarcinoma cell line that produces CEA.     

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     

Inserting chromosome 18 into pancreatic cancer cells switches them to a dormant metastatic phenotype.

We demonstrated previously that restoration of chromosome 18 suppressed growth of pancreatic cancer cells in vitro, as well as that of tumors inoculated into nude mice. We also demonstrated that loss of 18q was associated with poor prognosis. Hence there is the possibility that the 18q arm harbors a gene(s) implicated in tumor progression and/or metastasis. In this study, we evaluated the effect of restoring chromosome 18 on metastasis in a few human pancreatic cancer cell lines with and without inactivation of SMAD4. After microcell-mediated chromosome 18 transfer, hybrid cells showed more than a 10-fold weaker metastatic ability than corresponding parental cells; mice injected with 1.25 x 10(6)/250 micro l hybrid clones via tail vein had less than one-tenth of the number of macroscopic metastases in the lung when compared with the control cells. Microscopic examination confirmed the decrease in the number of metastatic lesions. After inoculation of hybrid cells, more than 80% of the high-power fields showed no micrometastases, contrasting with their abundance after using the parental cells. Hybrid cells restored maspin expression irrespective of SMAD4 status in corresponding parental cells. On the other hand, significantly lower vascular endothelial growth factor and matrix metalloproteinase 2 secretion was observed by measuring levels in the conditioned media (CM); the averages were 22% and 20%, respectively. Angiogenesis assays using in vivo Matrigel plugs demonstrated that less neovascularization was observed in nude mice with hybrid cells than with corresponding parental cells. When cells were treated with CM from hybrids, the migration of human umbilical vascular endothelial cells was decreased, but it was partially restored with anti-vascular endothelial growth factor neutralizing antibody, as compared with CM from parental cells. These data represent the first functional evidence suggesting that chromosome 18q encodes a gene that strongly suppresses metastatic activity, possibly through dormancy.     

Trisomy of rat chromosome 1 associated with mesothelial cell transformation.

Identification of specific chromosomal aberrations in transformed mesothelial cells is an important step in elucidating the mechanism of transformation of these cells which are targets for occupational and environmental carcinogens, such as asbestos fibers. Cytogenetic analysis of normal rat mesothelial cell lines revealed that at late passage (p20-p34), trisomy of chromosome 1 was present in greater than 80% of the cells in four spontaneously immortalized lines examined, whereas at early passage (p8-p10), only 15-44% of the cells had trisomy 1. Trisomy of chromosome 1 had increased in the population as a function of passage, suggesting that cells with trisomy 1 had a selective growth advantage under in vitro culture conditions and that this alteration was associated with transformation. A commercially available rat mesothelial cell line (4/4 RM4, ATCC), was also found to have a duplication of a portion of the long arm of chromosome 1. To determine if chromosome 1 alterations have relevance to the transformed phenotype in vivo, a neoplastic cell line was established from a spontaneous rat mesothelioma. At passage 15, trisomy of chromosome 1 was observed in 26% of the metaphases in this line. However, when these cells were injected into nude mice, 99% of the cells from the resulting tumor contained an additional copy of chromosome 1. Therefore, trisomy 1 also conferred a selective growth advantage in vivo and/or was associated with the malignant subpopulation in the tumor derived cell line. These studies suggest that chromosome 1 contains a gene(s) involved in transformation of rat mesothelial cells.     

Loss of heterozygosity for the short arm of chromosome 11 (11p15) in human milk epithelial cells immortalized by microinjection of SV40 DNA.

We have developed, by microinjection of SV40 DNA into human milk epithelial cells, a new mammary cell line, Hu-MI, which exhibits the phenotype of luminal cells or so-called "breast cancer precursor cells." This cell line retains the phenotype of primary cells as demonstrated by the expression of keratins 18 and 19 and of polymorphic epithelial mucins. However, the cells do not grow in agar after more than 80 passages, nor do they form tumors in nude mice. Established cells contain 2 copies of SV40 DNA integrated into the cellular genome and up to 14 copies of free SV40 DNA. A deletion of the short arm of chromosome 11 (11p15) including the c-Ha-ras and the beta-globin genes was found in the immortalized cells when the DNA from these cells was compared to the DNA from peripheral blood mononuclear cells obtained from the same donor. In addition, this cell line showed a good transfection efficiency for other DNA sequences using classical transfection and selection techniques with a neomycin resistance gene (pKOneo). Selective microinjection of DNA into tumor precursor cells may prove useful for the study of the molecular mechanisms involved in breast carcinogenesis. The possible significance of the loss of 11p13-15 in malignant progression of breast cancer is discussed.     

Homogeneously staining region in a retinoblastoma cell line: relevance to tumor initiation and progression

A human retinoblastoma cell line was found to contain a homogeneously staining region (HSR) on chromosome 1 (at 1p34). An HSR had previously been identified at the same site in a human neuroblastoma cell line. Of the original retinoblastoma line, a subpopulation was found which did not contain the 1pHSR but did contain a 3p+ chromosome in which the additional segment resembled a small HSR. The 3p+ was most likely the result of the translocation between the 1pHSR and the short arm of a chromosome 3. Preliminary results also indicate that the retinoblastoma cells with the 1pHSR produced tumors in athymic nu/nu mice in an average of 28 days, while identical numbers of the retinoblastoma cells with the 3p+ (probable HSR) produced tumors in an average of 75 days.     

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.     

Functional evidence for the presence of tumor suppressor gene on chromosome 10p15 in human prostate cancers

Loss of heterozygosity on chromosome 10p was observed frequently in human prostate cancers. Studies have demonstrated that the introduction of the short arm of human chromosome 10 into a human prostate cancer cell line, PPC-1, by microcell-mediated chromosome transfer (MMCT), suppressed the malignant phenotype, suggesting the presence of a prostate tumor suppressor gene(s) within a region of 17 cM at distal 10p. To narrow down the candidate region harboring the tumor suppressor gene, a series of 10p fragments were transferred into PPC-1 cells by MMCT using a panel of hamster-human hybrid cells containing various portions of 10p. Four of the six hybrid cells obtained showed decreased tumorigenicity when injected subcutaneously into athymic nude mice. Tumors developed only at six of 40 injection sites for these four hybrid cells. In contrast, the other two hybrid cells, as well as parental PPC-1 cells, were judged to be fully tumorigenic because tumors appeared at a total 26 of 32 sites for the two hybrid cells and 15 of 16 sites for PPC-1. Allelotyping of 10p combined with fluorescence in situ hybridization in these hybrid cells suggested that a prostate tumor suppressor gene was located within a fragment of approximately 1.2 Mb flanked by D10S1172 and D10S226 on 10p15.1.     

利用荧光素酶标记的肿瘤细胞建立活体成像动物模型

目的建立稳定表达荧光素酶的人乳腺癌细胞株并构建适用于小动物活体成像系统观察的裸鼠皮下移植瘤模型。方法采用脂质体将携带荧光素酶基因的质粒转染到人乳腺癌细胞株MCF-7中,G418筛选出稳定表达荧光素酶的单克隆细胞株。扩增后接种于裸鼠,建立裸鼠皮下移植瘤模型,通过活体动物成像系统监测肿瘤的生长过程。结果获得了高水平稳定表达荧光素酶的乳腺癌单克隆细胞株,该单克隆细胞株与母细胞系MCF-7具有相似的生长特性。将稳定表达荧光素酶的克隆接种于裸鼠皮下可成瘤,小动物活体成像系统能准确监测肿瘤细胞在体内的生长过程。结论成功建立了稳定表达荧光素酶的乳腺癌单克隆细胞株。采用活体动物成像系统构建的裸鼠皮下移植瘤模型是拓展肿瘤体内生长、转移及治疗相关研究的理想模型。     

Establishment of a new human pancreatic cancer cell line, NOR-P1, with high angiogenic activity and metastatic potential

We present here a new cell line, NOR-P1, established from a metastatic subcutaneous tumor of a patient with pancreatic cancer. The cells show rapid growth in culture with a doubling time of 16 h and high migration activity. Genetic and molecular analyses revealed high telomerase activity and a mutation in the K-ras oncogene. Of particular interest, the cells express markedly elevated mRNA levels of angiogenic factors, vascular endothelial growth factor and platelet-derived growth factor, as well as other tumor growth-related factors. Subcutaneous transplantation of the NOR-P1 cells into nude mice formed solid, hemorrhagic tumors which were histologically diagnosed as adenocarcinoma with dense blood vessels and severe extravasation of blood. Furthermore, when NOR-P1 cell suspension was injected directly into the pancreas of nude mice, the cells grew rapidly to form intra-pancreatic tumors associated with liver metastases and peritoneal dissemination that resulted in cachexia and subsequent death. These properties suggest that NOR-P1 is an aggressive pancreatic cancer cell line with a high metastatic potential and may serve as a useful experimental model for studying tumor angiogenesis and metastasis of pancreatic cancer.     

Tumor-necrosis-factor and cell-death in tumors (review)

The prognosis of pancreatic cystadenocarcinoma is known to be relatively favorable, unlike poor prognosis of the majority of pancreatic cancers. However, little is known about the pathogenesis and mode of progression of this cancer. We report on a newly established pancreatic cystadenocarcinoma cell line, OCUP-1. Its characteristics were compared to those of cells derived from pancreatic ductal cell carcinoma. OCUP-1 was established by successive culture of cancer cells obtained during an operation for pancreatic cystadenocarcinoma. This cell line demonstrated mono-layered proliferation and a doubling time of 80.3 h, which was 2.1 to 5.3 times longer than that of six ductal cell carcinoma-derived cell lines. Cell cycle time agreed with the doubling time. Tumors could be produced in all nude mice by inoculating cells from the 6 different ductal cell carcinoma derived cell lines without pre-treatment. However, with OCUP-1 cell line inoculation, the nude mice had to be pre-treated with asialo-GM1 - an inhibitor of natural killer cell activity - for tumors to be produced. Although the newly established OCUP-1 cell line demonstrated low proliferative activity, its genetic characteristics (point mutation at codon 12) were similar to those of other cells derived from ductal cell carcinomas. The OCUP-1 cell line may be used to investigate the pathogenesis and progression of pancreatic cystadenocarcinomas.     

Characterization of a newly established, TA-4-producing squamous carcinoma cell line derived from metastatic tongue carcinoma

A human squamous carcinoma cell line was established from the pleural effusion of a patient with recurrent squamous carcinoma of the tongue. The cell line, designated HST-I, has been passaged 82 times over a period of 4 years. The cells have been shown by light and electron microscopy to be of the squamous epithelial type. Immuno-histochemical staining was positive for keratin. When these cells were transplanted into athymic nude mice, tumors developed at the site of inoculation, which on histological examination were shown to be well-differentiated squamous carcinomas. Karyotypic analysis of cells from the cell line demonstrated an aneuploid human type with a modal chromosome number of 71, with both numerical and structural aberrations. HST-1 cells produce and secrete TA-4, a squamous-cell carcinoma-related antigen, in vitro in culture and in vivo in nude mice bearing the tumors produced by inoculation of cultured cells. Thus, the HST-1 cell line represents a new human tongue squamous carcinoma producing TA-4. This cell line appears useful for facilitating therapeutic investigations as well as biological studies on the association between cancerous growth and circulating TA-4 levels.     

Chromosome findings in human neuroblastomas xenografted in nude mice

Chromosomes were successfully studied in 8 of 9 human neuroblastomas (NBs) xenografted in nude mice. Structural abnormalities in the short arm of chromosome #1 were found in 6 of the 8 tumors; these included nonreciprocal translocations and simple deletions. The breakpoints were distributed between 1p11 and 1p34, and all of them had lost the terminal portion of 1p (1pter----1p34). Double minutes (DMs) and homogeneously staining regions (HSRs) were observed in 7 tumors; 6 had either DMs or HSRs, and one had some cells with DMs and other cells with HSR. Only one tumor had neither DMs, nor HSRs. Our study revealed that structural abnormalities in the NB xenografts were essentially the same as those in the NB cell lines and fresh tumors reported previously, and that DMs and HSRs were seen in most NB xenografts as frequently as in NB cell lines.     

Smad4基因沉默促进PanIN裸鼠移植瘤增殖和微血管形成的研究

背景与目的:由已建立的小鼠基因打靶模型证实,K-ras突变启动了胰腺癌前病变一胰腺腺管内上皮瘤(pancreatic intraepithelial neoplasia,PanIN),p53或p16失活均可单独促进小鼠PanIN发展为浸润性胰腺癌.作为人胰腺癌中另一失活频率高发的抑癌基因Smad4,其失活对PanIN的转化作用及是否可单独促进PanIN发展为胰腺癌目前仍不清楚.基于此目的,在已成功分离建立K-ras突变启动的PanIN细胞株基础上,本研究拟进一步应用RNA干扰技术沉默PanIN细胞株中内源性Smad4表达,以探讨siRNA干扰Smad4基因对PanIN细胞恶性转化作用.方法:构建Smad4基因沉默慢病毒质粒,筛选Smad4沉默稳转细胞并命名为PanIN-S细胞;分别用PanIN和PanIN-S细胞并采用皮下接种的方法,构建获得PanIN及PanIN-S细胞组裸鼠移植瘤模型(每组5只),2周后测定各组肿瘤体积和质量;应用免疫组织化学SP法检测并比较各组增殖细胞核抗原(PCNA)和CD31的表达及其差异.结果:成功构建了Smad4基因SiRNA体系;与未干扰PanIN细胞组相比,PanIN-S组裸鼠肿瘤体积和质量显著增加(P<0.05);组织病理学检查,符合胰腺癌(腺癌);免疫组织化学结果显示PCNA和CD31表达显著增高(P<0.05).结论:Smad4基因沉默可促使在K-ras突变基础上的小鼠PanIN细胞的恶性转化;裸鼠移植瘤中Smad4失活可显著促进小鼠移植瘤增殖和肿瘤微血管形成,这些可能是其致瘤恶性转化的重要作用机制.