Expression of HLA-DR antigens on tumour cells does not contribute to skin reactivity to autologous cholesteryl hemisuccinate (CHS)-treated tumour cells in patients with metastatic melanoma

Summary Skin tests with autologous cholesteryl hemisuccinate (CHS)-treated and untreated cells were performed in ten metastatic melanoma patients. In the majority of cases evident reaction was noted with CHS-treated cells (9/10) while the reaction with untreated cells was mostly negative (7/10). Tumour cell suspensions used for skin tests were characterized for reactivity with monoclonal antibody TAL 1B5 detecting the HLA-DR alpha chain. There were no differences between CHS-treated and untreated cells with respect to HLA-DR expression and no correlation was found between grade of skin reaction to CHS-treated cells and the proportion of HLA-DR positive cells in the injected cell sample.     

Modification of protoplast cell wall regeneration by membrane perturbation

Summary Protoplasts derived from cells ofBoergesenia forbesii regenerated aberrant cell walls when treated with cholesteryl hemisuccinate (CHS). Protoplasts treated with CHS, for a short period during the initial stages of cell wall regeneration, developed a patchwork cell wall, possessing regions devoid of cell wall. This effect was reversible, and treated cells ultimately developed a normal, confluent cell wall when removed from the CHS. Freeze fracture studies revealed that for CHS-treated cells, regions without microfibril impressions did possess intramembranous particles (IMP's) but that these regions contained small domains free of IMP's suggestive of lateral phase separation. The data implies that the physical characteristics of the plasma membrane lipid are important to the deposition of cell wall microfibrils during cell wall regeneration. This effect may be attributed to altered lipid-protein interactions, modified membrane fusion characteristics, or altered membrane flow.     

Involvement of lipid peroxidation and organic peroxides in UVA-induced matrix metalloproteinase-1 expression

Ultraviolet A (UVA) irradiation causes human skin aging and skin cancer at least partially through the activation of matrix metalloproteinases (MMPs). MMP-1, the interstitial collagenase, is responsible for the degradation of collagen and is involved in tumor progression in human skin. The present study uses human skin fibroblast cells (FEK4) to investigate the involvement of lipid peroxidation and the role of peroxides as possible mediators in MMP-1 activation by UVA. Preincubation with the antioxidants butylated hydroxytoluene and Trolox reduced UVA-dependent MMP-1 upregulation, suggesting that peroxidation of membrane lipids is involved. Blocking the iron-driven generation of lipid peroxides and hydroxyl radicals by different iron chelators led to a decrease in UVA-induced MMP-1 mRNA accumulation. Moreover, modulation of glutathione peroxidase activity by use of the specific inhibitor mercaptosuccinate (MS) or by the depletion of glutathione (using buthionine-S, R-sulfoximine, BSO), enhanced the UVA-dependent MMP-1 response. Finally, UVA irradiation generated a significant increase in intracellular peroxide levels which is augmented by pretreatment of the cells with BSO or MS. Our results demonstrate that lipid peroxidation and the production of peroxides are important events in the signalling pathway of MMP-1 activation by UVA.     

Skin tests with autologous cholesteryl hemisuccinate treated tumour cells in cancer patients

Summary Skin tests with autologous irradiated tumour cells were performed in 20 malignant melanoma, 7 breast and 6 ovarian cancer patients. In the majority of cases evident reaction was noted with cholesteryl hemisuccinate (CHS)-treated cells while the reaction with untreated cells was mostly negative.No correlation was found between this reactivity and the ability of patients to be sensitized to DNCB and to their reactivity to PPD. No correlation was found between reactivity to CHS-treated tumour cells and the stage and course of the disease.     

Incorporation of exogenous lipids modulates insulin signaling in the hepatoma cell line, HepG2.

The lipid content of cultured cells can be experimentally modified by supplementing the culture medium with specific lipids or by the use of phospholipases. In the case of the insulin receptor, these methods have contributed to a better understanding of lipid disorder-related diseases. Previously, our laboratory demonstrated that experimental modification of the cellular lipid composition of an insulin-sensitive rat hepatoma cell line (ZHC) resulted in an alteration in insulin receptor binding and biological action (Bruneau et al., Biochim. Biophys. Acta 928 (1987) 287-296/297-304). In this paper, we have examined the effects of lipid modification in another hepatoma cell line, HepG2. Exogenous linoleic acid (LA, n-6), eicosapentaenoic acid (EPA, n-3) or hemisuccinate of cholesterol (CHS) was added to HepG2 cells, to create a cellular model in which membrane composition was modified. In this model, we have shown that: (1) lipids were incorporated in treated HepG2 cells, but redistributed differently when compared to treated ZHC cells; (2) that insulin signaling events, such as insulin receptor autophosphorylation and the phosphorylation of the major insulin receptor substrate (IRS-1) were altered in response to the addition of membrane lipids or cholesterol derived components; and (3) different lipids affected insulin receptor signaling differently. We have also shown that the loss of insulin receptor autophosphorylation in CHS-treated cells can be correlated with a decreased sensitivity to insulin. Overall, the results suggest that the lipid environment of the insulin receptor may play an important role in insulin signal transduction.     

Epithelial-Stromal Interactions in Human Breast Cancer: Effects on Adhesion,Plasma Membrane Fluidity and Migration Speed and Directness

Interactions occurring between malignant cells and the stromal microenvironment heavily influence tumor progression. We investigated whether this cross-talk affects some molecular and functional aspects specifically correlated with the invasive phenotype of breast tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration) by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer-associated fibroblasts, CAFs) in 2D or 3D (nodules) cultures. Confocal immunofluorescence analysis of the epithelial adhesion molecule E-cadherin on frozen nodule sections demonstrated that NFs and CAFs, respectively, induced or inhibited its expression in MCF-7 cells. An increase in the mesenchymal adhesion protein N-cadherin was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of cancer cells. CAFs, in turn, promoted N-cadherin up-regulation in MDA-MB-231 cells and its de novo expression in MCF-7 cells. Beyond promotion of “cadherin switching”, another sign of the CAF-triggered epithelial-mesenchymal transition (EMT) was the induction of vimentin expression in MCF-7 cells. Plasma membrane labeling of monolayer cultures with the fluorescent probe Laurdan showed an enhancement of the membrane fluidity in cancer cells co-cultured with NFs or CAFs. An increase in lipid packing density of fibroblast membranes was promoted by MCF-7 cells. Time-lapsed cell tracking analysis of mammary cancer cells co-cultured with NFs or CAFs revealed an enhancement of tumor cell migration velocity, even with a marked increase in the directness induced by CAFs.Our results demonstrate a reciprocal influence of mammary cancer and fibroblasts on various adhesiveness/invasiveness features. Notably, CAFs'' ability to promote EMT, reduction of cell adhesion, increase in membrane fluidity, and migration velocity and directness in mammary cancer cells can be viewed as an overall progression- and invasion-promoting effect.     

Differential targets and subcellular localization of antitumor alkyl-lysophospholipid in leukemic versus solid tumor cells

Synthetic alkyl-lysophospholipids represent a family of promising anticancer drugs that induce apoptosis in a variety of tumor cells. Here we have found a differential subcellular distribution of the alkyl-lysophospholipid edelfosine in leukemic and solid tumor cells that leads to distinct anticancer responses. Edelfosine induced rapid apoptosis in human leukemic cells, including acute T-cell leukemia Jurkat and Peer cells, but promoted a late apoptotic response, preceded by G(2)/M arrest, in human solid tumor cells such as cervix epitheloid carcinoma HeLa cells and lung carcinoma A549 cells. c-Jun amino-terminal kinase (JNK) and caspase-3 were accordingly activated at earlier times in edelfosine-treated Jurkat cells as compared with drug-treated HeLa cells. Both leukemic and solid tumor cells took up this alkyl-lysophospholipid and expressed the two putative edelfosine targets, namely cell surface Fas death receptor (also known as APO-1 or CD95) and endoplasmic reticulum CTP: phosphocholine cytidylyltransferase. However, edelfosine was mainly located to plasma membrane lipid rafts in Jurkat and Peer leukemic cells and to endoplasmic reticulum in solid tumor HeLa and A549 cells. Edelfosine induced translocation of Fas, Fas-associated death domain-containing protein, and JNK into membrane rafts in Jurkat cells, but not in HeLa cells. In contrast, edelfosine inhibited phosphatidylcholine biosynthesis in both HeLa and A549 cells, but not in Jurkat or Peer leukemic cells, before the triggering of apoptosis. These data indicate that edelfosine targets two different subcellular structures in a cell type-dependent manner, namely cell surface lipid rafts in leukemic cells and endoplasmic reticulum in solid tumor cells.     

HAO2 inhibits malignancy of clear cell renal cell carcinoma by promoting lipid catabolic process

Hydroxy acid oxidase 2 (HAO2) has been reported to inhibit tumor progression through metabolic pathway. The current study was designed to evaluate the prognostic significance and probable mechanism of HAO2 in patients with clear cell renal cell carcinoma (ccRCC). The study screened The Cancer Genome Atlas Kidney Clear Cell Carcinoma (TCGA-KIRC) database for patients with ccRCC having complete clinical information and HAO2 expression. Low HAO2 was associated with shorter overall survival (OS) and shorter disease-free survival (DFS). Gene set enrichment analysis (GSEA) showed HAO2 was associated with neutral lipid catabolic process, metabolic process, lipid oxidation, epithelial–mesenchymal transition (EMT), and Kirsten rat sarcoma viral oncogene signaling (KRAS). Western blot analysis and immunohistochemistry analysis checked HAO2 expression in ccRCC cancer tissues, normal tissues, and renal cancer cell lines. HAO2 was downregulated in ccRCC cancer tissues and ccRCC cell lines when compared with their control group. Overexpression of HAO2 by plasmid promoted lipid catabolic process, eliminated lipid accumulation, inhibited KRAS expression, controlled the proliferation, migration, and invasion activity of ccRCC tumor cells. Our results indicated that HAO2 inhibits malignancy ccRCC by promoting lipid catabolic process, HAO2 could be an effective molecular marker and treatment for ccRCC.     

Active specific immunotherapy with an autologous tumor cell vaccine in patients with resected non-small cell lung cancer

Eighteen postoperative patients with non-small cell lung cancer were actively immunized with a vaccine that included autologous cryopreserved irradiated tumor cells admixed with bacillus Calmette-Guerin. Patients received three weekly intradermal immunizations beginning 1-3 months after surgery (15 patients) or after completion of postoperative radiotherapy (3 patients). There was marked heterogeneity in the relative proportion of tumor cells versus host infiltrating cells within individual vaccines (range of percent tumor cells 7-75%). Five patients exhibited positive delayed cutaneous skin test reactivity (DCR) to autologous irradiated tumor cells prior to immunization, whereas 8 of 13 converted from skin test negative to positive. There were no correlations between DCR reactivity and in vitro lymphoproliferative responses to autologous tumor cells or to clinical outcomes, i.e., freedom from relapse. Possible explanations for the heterogeneity of the lung cancer vaccine and approaches for improving its immunogenicity are discussed.     

Virus-augmented delayed hypersensitivity skin tests in gynecological malignancies

Summary Cultured human tumor cells of various histologic origins were infected with PR8/A/34 influenza virus. Nonviable crude membrane extracts were derived from the infected and uninfected cells. The extracts were coded and tested for their ability to produce delayed hypersensitivity skin reactions (DHSR) in allogeneic patients with squamous uterine cervical carcinoma, epithelial ovarian carcinoma, and malignant melanoma. Augmented antigen sensitivity to the virus-modified extracts compared with virus alone or to the unmodified extracts was observed in all patient groups. There was insufficient specificity to delineate a response by individual tumor type and related tumor extract, but some of the observed responses suggested tumor or organ site associations. Cervical carcinoma patients reacted more frequently to the virus-modified cervix extract, which also produced a high frequency of response in patients with ovarian carcinoma and melanoma. Ovarian carcinoma patients demonstrated increased sensitivity to both virus-modified ovarian carcinoma extracts, although 14 of 21 patients also showed responsiveness to one of the unmodified ovarian extracts. Malignant melanoma patients showed increased sensitivity to all virus-modified extracts except one of two derived from the ovarian carcinoma, and demonstrated a significantly augmented response to the virus-modified melanoma extract when the response to this extract was compared with that in ovarian carcinoma patients.The augmented reactions appear to be due to an association of the PR8 virus and as yet undetermined cellular components rather than to the virus alone. The possible involvement of tumor-associated determinants and the clinical significance of this phenomenon require further investigation.     

Fibroblasts prepared from different types of malignant tumors stimulate expression of luminal marker keratin 8 in the EM-G3 breast cancer cell line

It is widely recognized that stromal fibroblasts significantly influence biological properties of multiple tumors including breast cancer. However, these epithelial–mesenchymal interactions seem to be essential in tumor biology and it is not fully clear whether this interaction is tumor type-specific or has a more general non-specific character. To elucidate this question, we tested the effect of cancer-associated fibroblasts (CAFs) isolated from different types of tumors (breast cancer skin metastasis, cutaneous basal cell carcinoma and melanoma, squamous cell carcinoma arising from oral cavity mucous membrane) on the EM-G3 breast cancer cell line. The results were compared with control experiments using normal human dermal fibroblasts, 3T3 mouse fibroblasts, and 3T3 fibroblasts influenced by the fibroblasts prepared from the basal cell carcinoma. Our results demonstrated that expression of luminal marker keratin 8 was influenced only by CAFs prepared from any tested tumors. In contrast, all tested types of fibroblasts showed a strong stimulatory effect on the expression of basal/myoepithelial marker keratin 14. The CAFs also elevated the number of cells with positivity for both keratins 8 and 14 that are similar to ductal originated precursor cells. The expression of proliferation marker Ki67 was not influenced by any of the tested fibroblasts. In conclusion, our data indicate that CAFs are able to influence the phenotype of a breast cancer cell line and this effect is based on a tumor type-unspecific mechanism. Finally, a clear functional difference between normal and CAFs was demonstrated.     

In vivo studies on the uptake of radiolabelled antibodies by colorectal and gastric carcinoma xenografts

Summary Subjection of EL-4-leukemia cells to hydrostatic pressure of 1200–1500 atm for 15 min increased their weak basal immunogenicity to a potent practical level. Injection of such pressure-treated and irradiated EL-4 cells into syngeneic naive C57Bl/6 mice significantly delayed tumor development and increased survival after subsequent challenge with untreated EL-4 cells. Application of pressure of 1500 atm for a longer period of time (e.g., 120 min) resulted in cell death and a smaller increase in tumor immunogenicity which could be partially accounted for by passive shedding of membrane material. Unlike previously studied tumor cells, incorporation of cholesteryl hemisuccinate (CHS) into the plasma membrane of EL-4 cells increased their apparent tumor immunogenicity only slightly. In addition, isolated EL-4 plasma membranes, untreated, CHS-treated or pressure-treated, as well as the material shed thereof by hydrostatic pressure, were all of weak immunogenicity.Modulation in the projection of surface antigens upon pressure treatment could account for the observed increase in tumor immunogenicity and was monitored via the Thy 1.2 antigen. Fluorescence cell sorting analysis indicated that upon application of 1500 atm for different periods of time the projection of Thy 1.2 progressively and irreversibly increased to a maximal level of about 140% at 15 min. At longer pressurizations the availability of Thy 1.2 to antibody binding decreased sharply to levels below that of the untreated cells. It is suggested that pressure promotion of tumor immunogenicity is induced by changes in projection and surface distribution of the relevant antigens.Supported by a grant from the Institut National de lo Sante et de la Recherche Medicale FRANCE.     

弱碱性消癌液抑制乳腺癌细胞生长、转移并介导其消亡

为了研究新的肿瘤治疗方法,设计了1种弱碱性消癌液（weak alkaline cancer-eliminating liquid,WACEL）,测试WACEL是否抑制癌细胞的生长转移及消亡.在体外,检测WACEL对3种细胞株,即子宫颈鳞癌细胞SiHa、非小细胞肺鳞癌细胞H1299、人乳腺癌细胞MDA-MB-231的生长增殖、细胞周期、细胞凋亡、侵袭转移的影响. 在体内,建立人乳腺癌裸鼠颈背皮下移植瘤模型,观察WACEL对裸鼠皮下肿瘤生长转移的作用. 研究结果发现,WACEL明显抑制3种细胞系的生长增殖,G2/M期细胞增多,出现G2/M期阻滞,阻止细胞周期的进程. 细胞形态呈圆状,细胞核浓缩,caspase-3活性检测增加,线粒体的膜电位降低,细胞凋亡;活细胞数目减少,细胞膜破裂,发生消亡现象,癌细胞迁移明显减少（P﹤0.001）.目标基因SCCA1、cyclinB1、MMP-2以及MMP-9 的mRNA表达水平下调,caspase-3的mRNA表达水平上调;SCCA1、cyclinB1和MMP-2蛋白表达下调,caspase-3蛋白表达上调.体内动物实验发现,处理组的肿瘤生长较对照组明显缓慢,肺组织HE染色未见明显癌细胞转移,而对照组可见癌细胞转移. WACEL能抑制乳腺癌细胞的生长、转移并介导其消亡.本研究系统分析WACEL与癌细胞本身及其pH微环境之间的相互作用机制,发现其改变癌细胞所处的微环境的重要性.     

Regulation of lipid oxidation and structural state of the nuclear membrane after the irradiation of tumor-bearing animals

Changes of antioxidative activity (AOA), lipid composition and microviscosity of different membrane regions in tumor cell nuclei and in the liver of tumor-host with Ehrlich ascite carcinoma (EAC) after irradiation were studied. On the basis of the obtained data the analysis of the control system of lipid oxidation in the membrane was carried out. This control system involves a relationship between AOA changes, lipid composition, their oxidative ability and the nuclear membrane structure. It was shown that after irradiation the control system in the nuclei of tumor cells had the same state as before irradiation and was different from the normal one. The control system in the nuclei of tumor-host liver after irradiation starts to work in a regime which is characteristic of irradiated cells. It was shown that the principle difference in the control system functioning in tumor and tumor-host nuclei disappeared after irradiation.     

Microencapsulation [corrected] of tumor cells and assay for selecting anticancer drugs

A microencapsulation of living tumor cells by an improved membrane and droplet forming technique was established in our laboratory. This semipermeable microencapsulating membrane was impermeable to serum albumins (M.W. 66,000 or 45,000) and human hemoglobin (M.W. 64,000), but permitted passage of low molecular weight substances (alpha-Lactalbumin, or Trypsinogen; M.W. 14,200 or 24,000). The in vivo results showed that microencapsulated tumor cell lines (KB, human oral epidermoid cell; P-388 lymphocytic leukemia; GBM 8401/TSGH, glioma) and human colorectal carcinoma cells grew and proliferated exponentially within twenty days. The in vivo growth exhibited better than that in vitro. Histological and morphological findings of these four different kinds of tumor cells are similar to those of original tumor cells. Treatment of the microencapsulated tumor cells (MTC) with cytotoxic drugs (adriamycin, 5-fluorouracil and cyclophosphamide) in vitro showed no significant difference in percent inhibition (p greater than 0.05) between the encapsulated and non-encapsulated cells. The in vivo data indicated that different anti-cancer drugs had different inhibition effects. The results showed that the MTC model was useful for screening an appropriate cytotoxic drug and could be applied to clinical medicine in the near future.     

Effects of inhalation anesthetics halothane, sevoflurane, and isoflurane on human cell lines

Cytotoxic and antiproliferative effects of halothane, isoflurane, and sevoflurane in anesthetic doses on human colon carcinoma (Caco-2), larynx carcinoma (HEp-2), pancreatic carcinoma cells (MIA PaCa-2), poorly differentiated cells from lymph node metastasis of colon carcinoma (SW-620), and normal fibroblasts were investigated. Cells were exposed to anesthetic gas mixture consisting of O(2): N2O (35:60 vol.%), halothane (1.5 vol.%) or isoflurane (2.0 vol.%) or sevoflurane (3.0 vol.%), and CO(2) (5 vol.%), for 2, 4, and 6 h. Cytotoxicity of anesthetics was analyzed by validated tetrazolium dye assay MTT test. All anesthetics expressed cytotoxic effects on treated tumor cells in time and cell line dependent manner. Growth suppression in cells exposed to halothane was enhanced in HEp-2 (to 67.7%), Caco-2 (to 76.3%), and SW620 cells (to 80.9%), and was minimal in normal fibroblasts (to 89.4%). Antiproliferative activity of halothane was measured via radioactive precursors incorporation assay. In Caco-2 cells treated by halothane, decrease in DNA synthesis (52.4%, p=0.001), RNA synthesis (39.2%, p<0.001), and protein synthesis (19.2%, p=0.004) was observed. In HEp-2 cells, DNA and RNA syntheses were decreased to 72.5% and 79.9%, whereas protein synthesis was 14.0% of control (p<0.001). In SW620 cells, protein synthesis after 4 h was 24.4% (p=0.007). A DNA fragmentation was observed in Caco-2 and MIA PaCa-2 cells. Exposition of phosphatidylserine on outer lipid bilayer plasma membrane of tumor cell treated by halothane proved apoptosis as mode of cell death.     

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.     

Liquid-based cytology findings of glassy cell carcinoma of the cervix. Report of a case with histologic correlation and molecular analysis

BACKGROUND: Glassy cell carcinoma is a rare form of poorly differentiated carcinoma of the cervix with no obvious squamous or glandular differentiation. Its liquid-based cytology findings have not been described before. CASE: A 46-year-old Filipina presented with vaginal bleeding due to a bulky cervical tumor. The liquid-based cytology preparation was of moderate cellularity and contained small clusters of polygonal to elongated tumor cells admixed with amphophilic, granular, necrotic debris. The malignant cells possessed round to oval nuclei; a thin nuclear membrane; finely dispersed chromatin; prominent, solitary nucleoli; abundant, cyanophilic cytoplasm; and discrete cell borders. Occasional tumor cells showed phagocytosis of polymorphs. The background contained a mixed population of inflammatory cells. Eosinophils, though present, were not readily identified in the cytologic specimen. There was no evidence of dyskeratosis, cytoplasmic vacuolation or koilocytosis. Histologic and ultrastructural examination of the tumor biopsy showed classic features of glassy cell carcinoma. Molecular analysis using polymerase chain reaction and restriction fragment length polymorphism revealed the presence of human papillomavirus (HPV) DNA in the liquid-based cytology sample. The HPV genotype, however, did not belong to any of the commonly encountered prototypes. CONCLUSION: Glassy cell carcinoma of the cervix may show distinct, though subtle, cytomorphologic features in liquid-based preparations. The findings, however, are slightly different from those in conventional cervical smears. Awareness of this rare entity is important, as glassy cell carcinoma is often associated with more aggressive clinical behavior.