Low-dose recombinant interleukin-2 and low-dose cyclophosphamide in metastatic breast cancer

Summary We undertook a preliminary study to examine the response rate of recombinant interleukin-2 (rIL-2) in patients with advanced measurable breast cancer, in a phase II clinical trial. The regimen we utilized was designed to allow outpatient administration. A treatment cycle consisted of low-dose cyclophosphamide (350 mg/m²) given on day -3 followed by the bolus administration of rIL-2 (3.6 × 10⁶ Cetus units/m²) on days 1–5, and 8–12. Toxicity was significant but acceptable. One partial remission was seen in 13 evaluable patients. In 2 additional patients clear evidence of an antitumor response was observed. The study was terminated prematurely owing to a shortage of rIL-2. Additional evaluation of rIL-2 in breast cancer appears warranted.     

Transient transfection of epidermal growth factor receptor gene into MCF7 breast ductal carcinoma cell line

Epidermal growth factor receptor (EGFR) is activated by autocrine growth factors in many types of tumours, including breast tumours. This receptor has been linked to a poor prognosis in breast cancer and may promote proliferation, migration, invasion, and cell survival as well as inhibition of apoptosis. Human breast ductal carcinoma MCF7 cells were transfected using FuGENE 6 with 1 microg of pcDNA3-EGFR containing the full-length human EGFR promoter or 1 microg of the vectors alone (pcDNA3). The transfected cells were transferred into a 25-cm2 flask containing growth medium and G418. Confluent cultures were lysed, total protein levels measured and electrophoresed. The electrophoresed samples were transferred to nitrocellulose and incubated overnight at 4 degrees C with either anti-EGFR or anti-phospho-ERK and immunoreactive bands were visualized using HRP-linked secondary antibody. We created a model system of EGFR overexpression in MCF7 clones with stably transfected pcDNA3/EGFR plasmid. These cells have been shown to promote substantial phosphorylation of both ERK1 and ERK2. The high level of EGFR and ERK1/2 phosphorylation was not seen in the pcDNA3 vector control cells or in non-transfected cells. In this article we describe successful transient transfection experiments on MCF7 cells using the FuGENE 6 Transfection Reagent. The overexpression of EGFR could be a mediated stress response and a survival signal that involves ERK1 and ERK2 phosphorylation.     

Establishment and characterization of a human thyroid carcinoma cell line (HOTHC) producing colony stimulating factor

A cell line designated HOTHC was established from an anaplastic carcinoma (giant cell type) of the thyroid gland of 80-year-old woman. The HOTHC line grew rapidly in multilayer without contact inhibition, and more than 120 serial passages were made within 27 months. The cells were spindle or polygonal in shape and revealed neoplastic and pleomorphic features. These cells were characterized as containing coloid droplets and poorly developed rough-endoplasmic reticulum in the cytoplasm. Doubling time was about 24 hours and plating efficiency was about 70%. The karyotype exhibits hyperploidy and marker chromosomes, and the modal chromosome number ranged between 77-90. The HOTHC cells were transplanted into the subcutis of BALB/c nude mice and produced anaplatic carcinomas (giant cell type) resembling the original tumor. The HOTHC cells produced colony stimulating factor (CSF) and caused granulocytosis in the mice.     

Establishment and characterization of a new human cell line (EJ) derived from endometrial carcinoma

We present a new cell line, EJ established from an invasive endometrioid adenocarcinoma of the uterine corpus in a 56-year-old patient. The cells show rapid growth in culture with a doubling time of 16 h and high migration activity. Monolayer-cultured cells were polygonal in shape showing a tendency to pile up without contact inhibition. Subcutaneous transplantation of the EJ cells into nude mice formed solid tumors that were histologically diagnosed as adenocarcinoma, whereas no metastasis was observed. Cultured EJ cells produced tissue polypeptide antigen (IPA). Genetic and molecular analyses revealed high telomerase activity but not estrogen receptor alpha expression. Using the DNA sequencing technique, we have screened EJ cells for p53 mutation in exon 5 to 8 but no mutation of p53 was observed. This cell line appears to represent the development of a more malignant clone with divergent receptor function and growth behavior, and provides us with an interesting new tool for the study of tumorigenesis in the human endometrium.     

Establishment and characterization of a new human hepatocellular carcinoma cell line

Summary A human hepatocellular carcinoma cell line (FOCUS—Friendship of China and United States) was derived from a patient with primary hepatocellular carcinoma. This cell line has been in continuous culture over an 18-mo period. The morphological and ultrastructural features of FOCUS are consistent with its neoplastic hepatocellular orgin. FOCUS cells contain aspartate aminotransferase and glucose-6-phosphatase activity. In addition, α₁-antitrypsin, fibrinogen, alpha fetoprotein, and carcinoembryonic antigens were detectble in the cytoplasm of the cultured cells by immunochemical staining techniques. The karyotype of the FOCUS cell is human in origin and it contains human DNA sequences as detected by molecular hybridization analysis. The FOCUS cells do not show evidence of density-dependent inhibition of growth under confluent conditions. Repeated growth curves over an 18-mo period were identical, revealing a doubling time of 42 to 48 h. The malignant potential of FOCUS cells was further demonstrated by their ability to lead to gross tumor formation after subcutaneous infection into nude mice. From one of the solid tumors grown in nude mice, recultured cell lines have been established and found to have properties identical to the original FOCUS cell line. This FOCUS cell line represents an additional model for further investigation of tumor specific antigens and the relationship between hepatitis B virus (HBV) and hepatocellular carcinoma. Preliminary molecular characterization has indicated the existence of integrated HBV sequences within the FOCUS genome.     

Inhibition of tumor cell growth by a human B-cell line

An Adenocarcinoma cell line (Breast-M) and an Epstein-Barr virus (EBV)-infected B-cell line (Hairy-BM) were established from breast tumor tissue. The Hairy-BM was CD20⁺, CD25 (Tac)+ and surface immunoglobulin (sIg)+. Hairy-BM suppressed the in vitro proliferation of Breast-M in a time and a dose-dependent manner. The suppression was also found in 5 different human tumor targets showing tumor-Hairy-BM binding, but not; in 2 murine tumor targets showing no significant tumor-Hairy-BM binding. Lytic activity of Hairy-BM was found only against Breast-M.Abbreviations sIg Surface immunoglobulin - CTL Cytotoxic T-cells - NK Natural killer - IL2 Interleukin 2 - LAK Lymphokine activated killer - CSN Culture supernatant - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoolium bromide - PCR Polymerase chain reaction - TIL Tumor-infiltrating lymphocytes - HCL Hairy cell leukemia - TNF Tumor necrosis factor     

The expression of interleukin-6 by a rat macrophage-derived cell line

A stable rat macrophage-derived cell line (RMSV1) was established by transformation of primary peritoneal exudate cells with the SV40 virus. The RMSV1 cell line was used as a model to study the regulation of the interleukin-6 (IL6) gene expression in rate macrophages with respect to lipopolysaccharides (LPS), interleukin-1 (IL1) and glucocorticoids. The IL6 mRNA level in RMSV1 cell lines was induced 20-fold within 4 h by LPS, whereas IL1 had no effect. The glucocorticoids were able to inhibit completely the induction of the IL6 mRNA synthesis by LPS, indicating the negative regulation of the IL6 gene expression by glucocorticoids.     

Establishment and characterization of a cell line (SS78) from a human renal cell carcinoma

Summary A new cell line, SS78, was established from a primary renal cell carcinoma of a Caucasian male. The tissue was dispersed with collagenase, and viable cells were separated by flotation on a Ficoll-Hypaque gradient. In culture, the SS78 cells retained a distinct epithelial morphology, and no fibroblastlike cells were seen. The cultured cells were aneuploid with a modal chromosome number of 80 and had several marker chromosomes. Inoculation of the cultured cells into athymic nude mice caused tumors at the sites of inoculation. This research was supported in part by Grants CA 15972 and CA 14930 from the National Cancer Institute through the National Bladder Cancer Project and by the Medical Research Service of the Veterans Administration.     

Multiple drug-resistance in variant of a human non-small cell lung carcinoma cell line,DLKP-A

A 300-fold adriamycin resistant variant (DLKP-A) of the human lung squamous cell carcinoma line DLKP was established by stepwise selection in increasing concentrations of adriamycin. Different levels of cross-resistance were observed towards VP-16, VM-26, colchicine, vincristine and, somewhat unexpectedly, cis-platin. Resistance was stable for at least 3 months in culture in the absence of drug. P-glycoprotein overexpression was detected by immunofluorescence and Western Blotting, and a direct causal role for P-glycoprotein overexpression in the resistant phenotype was established by transfection with an mdr1 specific antisense oligonucleotide. A modified cryopreservation procedure was necessary for the resistant variant line. The resistant population displays clonal heterogeneity with respect to resistance level. A higher frequency of double minute chromosomes was observed in DLKP-A when compared with the parental cell line.Abbreviations ADR adriamycin - COLH colchicine - C-PT cis-platin - MDR multidrug resistance - NSCLC non-small cell lung carcinoma - VCR vincristine - VP-16 etoposide - VM-26 tenoposide     

一株新的人肾癌原代细胞系的建立及其生物学特性鉴定

目的:取肾癌病人标本建立一株新的人肾癌细胞系,初步对该细胞系进行鉴定,为进一步的肾癌基础研究提供实验模型.方法:2008-2009年间共采集20例肾癌新鲜手术标本,于手术后1 h内将每例标本切取四块0.5cm× 0.5 cm× 0.5 cm组织块,分别包埋于2只裸鼠的右后肢皮下及背部皮下,连续传代3次,取移植瘤体外培养,记录细胞株的生长曲线,测定细胞集落形成率,对细胞进行DNA含量测定,进行染色体分析及病理学检查.结果:其中1只裸鼠皮下移植瘤生长,继续传代,肿瘤生长速度明显加快.取移植瘤标本体外培养得到肾癌细胞系XJG-9201.形态结构,分化程度与原发瘤一致,染色体众数为65.细胞群体倍增时间为38.2h,细胞周期分析G1期62.7%,G2期11.2%,S1期25.3%,集落形成率为70%.结论:肾癌细胞系XJG-9201与原发癌保持相同的生物学特性,体外连续传代1年以上传115代.细胞形态不变,生长周期恒定,已成为一个稳定的细胞系.     

Establishment and characterization of a cell line derived from a human serous surface papillary carcinoma of the ovary

A novel serous surface papillary carcinoma of the ovary (SSPC) cell line, HYKSSPC, was established successfully. Carcinoma cells were obtained from ascitic fluid of a 60-year-old Japanese woman. The population doubling time was 51.4 h. A phase contrast micrograph showed a pavement stone-like arrangement without contact inhibition. The chromosome number showed a wide distribution of aneuploidy, and the mode was in 46-47. An immunocytochemical study showed that CA125, BerER4 and cytokeratin were positive and that CEA, calretinin and thrombomodulin were negative. This cell line preserved some characters of the adenocarcinoma while growing in vitro. A chemosensitivity test revealed that HYKSSPC cells were sensitive to CDDP (cis-platinum), 5-fluorouracil, mitomycin C, paclitaxel and irinotecan. To our knowledge, HYKSSPC is the first established cell line derived from SSPC, and it may offer some useful information for investigating this disease.     

Ovarian carcinoma cells inhibit T cell proliferation: suppression of IL-2 receptor beta and gamma expression and their JAK-STAT signaling pathway

Deficient T cell immune function and intracellular signaling in cancer patients may result from effects of tumors or their products on lymphocytes. Recently, it was demonstrated that several ovarian carcinoma cell lines could produce soluble factors that inhibited T cell proliferation. The aim of this study is to assess the effect of supernatants from 3 ovarian carcinoma cell lines (OVCAR3, CAOV3, SKOV3) on signal transduction elements that are linked to the IL-2R and its JAK-STAT pathway. A profound inhibition of proliferation, lower level of IFN-gamma and higher level of IL-10 gene expression were observed when CD8+ T cells were co-cultured with supernatants from 3 ovarian carcinoma cell lines. Cell cycle studies on inhibited CD8+ T cells showed most of them were growth arrested in G0/G1 phase. Western blot analysis showed that tumor supernatants suppressed expression of JAK3 and tyrosine phosphorylation of STAT5. JAK1 was not altered and the inhibition of STAT3 only appeared in OVCAR3 cells. Tumor supernatants also partially blocked induction of IL-2R beta and gamma chains expression. These findings suggest that ovarian carcinoma cells may suppress T cell proliferation through inhibition IL-2 dependent signaling pathways, which may be a mechanism of ovarian carcinoma induced immunosuppression.     

A new human hepatocellular carcinoma cell line (KYN-1) with a transformation to adenocarcinoma

Summary A human hepatocellular carcinoma (HCC) cell line (KYN-1) has been established from a resected HCC of a 58-yr-old Japanese, male patient with HCC. Original resected HCC was moderately differentiated and proliferated in a solid pattern with vague trabecular structure in part. This cell line has been maintained for 10 mo. through 50 passages. Morphological features of KYN-1 cells demonstrated one or more large, round-to-oval nuceli with prominent nucleoli and eosinophilic polygonal-to-spindle abundant cytoplasm. In addition, some of these cells contained mucicarmin-positive materials in the cytoplasm. The cells exhibited a typical epithelial feature with pavementlike cell arrangement, and lacked contact inhibition. The doubling times of the cells grown in a serum-containing and a serum-fre, medium were about 31 h and 10 to 11 d, respectively. Functonally, KYN-1 cells produced albumin, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin, β₂-microglobulin (BMG), and α₁-anti-trypsin (AAT). Positive reactions for albumin, AFP, CEA, and ferritin were identified in the cells by immunohistochemical techniques. Chromosome study revealed the chromosome number in a range from 61 to 74 without mode. The tumorigenicity of KYN-1 cells was identified by the tumor formation after subcutaneous inoculation of the cells into nude mice. The developed tumor showed compact growth of the tumor cells with gland formations containing mucicarmin-positive materials. Features of adenocarcinoma were identified by electron microscopy. The tumor cells were also identified to contain albumin, AFP, CEA, ferritin, and AAT by immunohistochemical techniques. AFP, CEA, and BMG were detected in the sera of nude mice. Thus, KYN-1 cells represented the morphologic features of adenocarcinoma, retaining some characteristics of original HCC. These findings suggest that KYN-1 is a new human HCC cell line with transformation to adenocarcinoma, which will provide useful information to clarify the histogenesis of combined hepatocellular and cholangiocellular carcinoma. This study was supported in part by the Sarah Cousins Fund.     

Sensitivity of human pancreatic carcinoma cell line (MIA PaCa-2) to Bisantrene and Theprubicin in vitro

Summary We tested the effect of Bisantrene (BS) and Theprubicin (THP-ADR) on cell growth of a human pancreatic carcinoma cell line (MIA PaCa-2). After 1 h exposure ID₅₀ of BS or THP-ADR was 3×10⁻⁷ and 5×10⁻⁸ M, respectively. Increasing the exposure time from 1 h to continuous exposure for 5d resulted in 11-fold decrease in ID₅₀ for BS and a 6-fold decrease for THP-ADR. Both drugs inhibited [¹⁴C]thymidine incorporation to the same extent and caused an accumulation of cells into G₂+M phase of the cell cycle. This work is supported by a gift, from Mr. Issam Fares and U.S. Public Health Service Grants AM 07114 and CA 19182.     

Establishment and characterization of a new human renal cell carcinoma cell line (KRC/Y)

Summary A new renal cell carcinoma (RCC) cell line (KRC/Y) has been established from a surgical specimen of a 41-yr-old Japanese female patient with RCC composed of both clear cells and granular cells. This cell line has been maintained for more than 15 mo. through 45 passages with a stable growth, KRC/Y cells have clear or eosinophilic polygonal cytoplasm and round to oval nuclei with one or two nucleoli, and proliferate in a pavementlike cell arrangement with a lack of contanct inhibition. By electron microscopy, these cells contain abundant fat droplets and glycogen granules or well-developed organells or both, which were also observed in the original tumor. The doubling time of these cells at the 15th passage was 73 h. The chromosome number was from 37 to 45 with a hypodiploid modal number of 42. Tumorigenicity was identified by tumor formation after subcutaneous injections of KRC/Y cells in nude mice, which showed close resemblance to the original tumor by light and electron microscope observations. This study was supported in part by Sarah Cousin Fund, Boston, MA.     

2-DE analysis of a new human cell line EM-G3 derived from breast cancer progenitor cells and comparison with normal mammary epithelial cells

We performed a 2-DE analysis of proteins of the newly established spontaneously immortalized clonal cell line EM-G3 derived from a primary lesion of infiltrating ductal breast carcinoma. EM-G3 cells may represent progenitors of the mammary epithelial cells spontaneously immortalized in early phase of cancerogenesis. We compared the protein profile of EM-G3 line with proteins from populations of normal mammary epithelial cells (NME), and determined the phenotype of both types of cells. NME cells are a mixture of both main cell types in breast epithelia, myoepithelial and luminal cells. The EM-G3 breast cancer cell line has a unique basal-like phenotype. We identified proteins that are differently expressed in these cells. Cytokeratin 16, cytokeratin 19, squamous cell carcinoma antigen 1, caphepsin B and caspase 14 were predominantly expressed by NME cells. Cytokeratin 13, isoelectric variant of annexin 5, isoelectric variant of chloride intracellular channel protein 1, glyoxalase 1 and glutamine synthetase were predominantly expressed by EM-G3 cells. The proteins up-regulated in EM-G3 cells may represent potential protein markers of mammary epithelial cells progenitors and may be important in early phase of carcinogenesis.     

白细胞介素-2受体在乳腺癌MCF-7细胞的表达

目的 :观察乳腺癌MCF 7细胞上白细胞介素 2受体 (IL 2R)α、β和γ链的表达、IL 2对MCF 7细胞增殖的作用及雌激素对三条链表达的影响。方法 :使用特异性IL 2R多克隆抗体以免疫细胞化学方法和流式免疫荧光法检测MCF 7细胞上IL 2R的表达 ,以MTT法及3 H TdR掺入法检测细胞增殖情况。结果 :MCF 7细胞上存在IL 2Rα、β、γ的免疫阳性物质 ,其中IL 2Rγ的表达要强于IL 2Rα、β的表达 ;10 -6mol/L浓度的雌二醇可促进IL 2Rα、β的阳性细胞数及IL 2Rγ的免疫阳性物质的含量 ;IL 2在 10 0U/ml至 10 0 0U/ml的浓度范围内可显著促进MCF 7细胞的增殖。结论 :MCF 7细胞上存在IL 2R且其表达受雌二醇的调节 ,IL 2可能通过IL 2R影响MCF 7细胞的增殖     

Human renal cell carcinoma: Establishment and characterization of a new cell line (OS-RC-2)

Summary A cell line, designated as OS-RC-2, has been established from a renal cell carcinoma in a 52-yr-old Japanese male patient and maintained for 23 mo. through 60 in vitro passages. The OS-RC-2 formed monolayers of polygonal epithelial cells and lacked contact inhibition. Doubling time of cells was about 60 h at the 30th passage. Electron microscopic findings indicated numerous long microvilli on the cell surface and many glycogen granules in the cytoplasm of this cell line, which are characteristic structures of renal cell carcinoma. Chromosomal analysis revealed that a small portion of this cell line had a hypodiploid modal number of 40 and a large portion had a hypotetraploid modal number of 75. The characteristics of the karyotype were one detected marker chromosome and the translocation between the Chromosomes 2 and 13. Cell line OS-RC-2 was serially transplantable into nude mice, and histopathological findings of heterotransplanted tumor showed a close similarity to those of the original tumor. Histocompatibility antigens of OS-RC-2 were HLA-A9, Bw52, which were identical to those of the peripheral blood lymphocytes of the patient.