bluuebunny/crossref_metadata_2025 huggingface dataset:
in vitro effects of recombinant human interferon gamma on human mesothelioma cell lines
Authors: Zeng L. , Buard A. , Saint‐Etienne L. , Jaurand M. C. , Bignon J. , Monnet I. , Brochard P. , Boutin C. , Fleury J.
Published: February 2007

Malignant mesothelioma is a tumor arising from serous surfaces and often related to asbestos exposure. Malignant mesothelioma is resistant to various forms of therapy. Radiotherapy, surgery or chemotherapy only slightly improve prognosis. IFN‐γ produces complete or partial responses in stage‐l patients with malignant mesothelioma. The in vitro biological effect of IFN‐γ on malignant mesothelioma cells remains poorly elucidated. In the present study, 32 well‐characterized human mesothelioma cell lines (HMCL) were treated with r‐hu IFN‐‐γ at 4 doses and cell growth was determined by a colorimetric method (MTT assay). Among the 32 HMCLs tested, 11 exhibited significant cell‐growth inhibition; 16 were insensitive to r‐hu IFN‐γ, and 5 were slightly inhibited. The sensitive cell lines were strongly inhibited by r‐hu IFN‐γ. Our results show that HMCL exhibit a large range of responses to r‐hu IFN‐γ, some of which can be compared with those obtained in vivo in humans.
Modulation of sensitivity of human ovarian cancer cells to cis‐diamminedichloroplatinum(II) by 12‐O‐tetradecanoylphorbol‐13‐acetate and D, L‐buthionine‐S, R‐sulphoximine
Authors: Hirata Junko , Kikuchi Yoshihiro , Kita Tsunekazu , Imaizumi Eiji , Tode Takehiko , Ishii Kenji , Kudoh Kazuya , Nagata Ichiro
Published: February 2007

The ability of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and D, L‐buthionine‐S, R‐sulphoximine (BSO) to modulate cisdiamminedichloroplatinum(11) (CDDP) sensitivity was investigated in human ovarian cancer cell lines sensitive (KF) or with intrinsic resistance (KK and MH) to CDDP. The KK and MH cell lines were derived from ascites of patients with clear‐cell carcinoma and serous cystadenocarcinoma of the ovary who both showed clinical resistance to CDDP. The CDDP IC50 value of KK and MH cells was about 4.6‐ and 10.2‐fold higher than that of KF cells. PKC activities in the cytosol and membrane of KK and MH cells were also about 4‐ to 5‐fold higher than those of KF cells. Proliferation of KF, KK and MH cells was inhibited in a dose‐dependent manner by TPA. The membrane PKC activities in the KF cells were rapidly activated and down‐regulated 24 hr after exposure to TPA, while those in the KK and MH cells were not down‐regulated even after exposure to TPA for 24 hr, suggesting that the membrane form of PKC may be involved in the intrinsic resistance. Continuous exposure to 10 nM TPA for 5 days significantly reduced the CDDP sensitivity of KF and KK cells, while exposure to 10 nM TPA for I hr significantly elevated that of KK and MH cells. Interestingly, I ‐hr exposure to I m̈M TPA induced CDDP‐resistance in KK cells. Such changes in CDDP sensitivity by TPA seemed to be linked with those of cellular PKC activity, i.e., when the CDDP sensitivity was reduced by TPA, the cellular PKC rose. When the CDDP sensitivity was increased, the cellular PKC decreased. In addition, glutathione (GSH) levels in KK and MH cells, whose proliferation was more markedly inhibited by BSO than the KF‐cell proliferation, were about 7.6‐ and 1.9‐fold higher than those in KF cells. The degree of depletion of GSH by BSO was more marked in the KK and MH cells than in the KF cells, subsequently resulting in a significant elevation of the CDDP sensitivity of KK and MH (but not KF) cells. Our results suggest that intrinsic resistance to CDDP can be reduced by TPA or BSO under some conditions.
Tobacco use and nasopharyngeal carcinoma in a cohort of us veterans
Authors: Chow Wong‐Ho , McLaughlin Joseph K. , Hrubec Zdenek , Nam Jun‐Mo , Blot William J.
Published: February 2007

The risk of nasopharyngeal carcinoma (NPC), a relatively rare neoplasm in the United States, was examined in relation to tobacco use in a cohort of nearly 250,000 US veterans whose mortality experience was followed for 26 years. A total of 48 NPC deaths were identified during the follow‐up period. Current smokers had a nearly 4‐fold increase in risk of NPC compared with non‐users of any tobacco, with risk increasing to 6.4 among those smoking more than 2 packs daily. After adjustment for age and number of cigarettes smoked, risks were inversely associated with age at starting to smoke, with the highest risk observed among those who started smoking before age 15, although no clear trend associated with duration of smoking was observed. Former cigarette smokers had a small excess risk of NPC, but no association was detected for cigar/pipe users. This study adds strong evidence to the increasing literature linking cigarette smoking to NPC.
Abnormalities affecting the APC and MCC tumour suppressor gene loci on chromosome 5q occur frequently in gastric cancer but not in pancreatic cancer
Authors: McKie Arthur B. , Lemoine Nicholas R. , Filipe M. Isabel
Published: February 2007

Abnormalities affecting tumour suppressor genes on chromosome 5q21 are increasingly recognised as important in the pathogenesis of a variety of human cancers, particularly of the gastrointestinal tract. We have examined a series of gastric and pancreatic cancers from European patients for loss of heterozygosity (LOH) of markers within and around the APC and MCC genes on chromosome 5q2t using restriction fragment length polymorphism and polymerase chain reaction techniques. We find that LOH of the APC and MCC genes is particularly frequent in gastric cancers of diffuse type, but very infrequent in pancreatic cancers. We have also used single‐strand conformational polymorphism to screen for abnormalities of the sequence of the APC and MCC genes in a panel of pancreatic cancer cell lines. Our results suggest that there are distinct differences in the molecular pathogenesis of gastric and pancreatic cancer and that abnormalities of APC and MCC may be involved particularly in the diffuse type of gastric cancer.
Moloney virus induction of T‐cell lymphomas in a plasmacytomagenic strain of Eμ‐V‐ABL transgenic mice
Authors: Haupt Ygal , Harris Alan W. , Adams Jerry M.
Published: February 2007

Although the v‐abl gene can provoke several types of lymphoid neoplasm, mice of a transgenic strain (Em̈‐v‐abl 40) in which lymphocytes are targeted for expression of v‐abl by a linked immunoglobulin enhancer (Em̈) spontaneously develop only plasmacytomas. To determine whether other lymphocytes of this strain were susceptible to transformation, and to identify genes that can collaborate with v‐abl in tumorigenesis, Em̈‐v‐abl 40 mice were subjected to insertional mutagenesis by neonatal infection with Moloney murine leukemia virus. Tumorigenesis was accelerated moderately, but nearly all the tumors were T lymphomas. The altered tumor type may reflect both the T‐cell tropism of Moloney virus and the higher level of Em̈‐v‐abl 40 expression found in T lymphocytes than in B lymphocytes. Insertion near the c‐myc, N‐myc or pim‐1 gene was observed in 42% of the induced tumors, indicating that each of these genes may collaborate with v‐abl in lymphomagenesis. Most of the accelerated tumors had a surprisingly low level of transgene expression. Thus, high expression of v‐abl may not be required for Moloney‐induced T lymphomagenesis.
Production of monoclonal antibodies to human estrogen‐receptor protein (ER) using recombinant ER (RER)
Authors: Saati Talal Al , Cohen‐Knafo Edith , Caverivière Paul , Delsol Georges , Clamens Simone , Faye Jean Charles , Prats Hervé , Bayard Francis , Coindre Jean Michel , Wafflart Jean
Published: February 2007

Two monoclonal antibodies (MAbs), IC5 and ID5, were produced using spleen cells from BALB/c mice immunized with recombinant estrogen‐receptor protein (RER). On immunoblotting, both MAbs reacted with the 67‐kDa polypeptide chain obtained by transformation of E. coli and transaction of COS cells with plasmid vectors expressing ER. The epitopes of both MAbs were in the N‐terminal domain (A/B region) of the receptor. In normal human tissues, IC5 and ID5 reacted with cells known to contain large amount of ER, such as cells of the mammary gland and the uterus. Staining was localized predominantly in nuclei with little or no cytoplasmic reactivity. IC5 and ID5 were unreactive with tissues usually considered to be negative for ER. The reactions of these 2 MAbs were further tested on different tumor types, using immunohistochemical (IHC) method on frozen sections. In breast cancer, a good correlation was found between the results obtained on frozen sections and those using the conventional radioligand dextran‐coated charcoal (DCC) assay. Immunostaining with IC5 and ID5 MAbs was also assessed on routinely processed paraffin sections using the antigen‐retrieval method. Staining was comparable to that obtained on frozen sections in virtually all the breast carcinomas. Negative reactions were consistently obtained with both antibodies on human neoplasms derived from other non‐estrogen‐dependent organs. IC5 and ID5 MAbs may thus be of value in routine diagnostic histopathology for assessment of the estrogen‐receptor content in human carcinomas.
A sub‐set of immediate early mRNAs induced by tumor necrosis factor‐α during cellular cytotoxic and non‐cytotoxic responses
Authors: Klefstrom Juha , Saksela Eero , Alitalo Kari , Koskinen Päivi J. , Jäättelä Marja , Bravo Rodrigo
Published: February 2007

TNF‐α is a multifunctional cytokine which is cytotoxic for some cell lines. In order to characterize the early genomic response to TNF‐α, we have analyzed the induction of a sub‐set of serum‐inducible immediate early genes in WEHI‐S and L929 fibrosarcoma cell lines, which are sensitive to TNF‐α, and in the 3T3‐L1 pre‐adipocytic cell line, which is resistant to TNF‐α cytotoxicity. Among 77 immediate early mRNAs screened by dot blot and/or Northern blot analyses, the expression of 23 mRNAs was found to be induced by TNF‐α. Ten of these mRNAs encode proteins known to function as pro‐inflammatory cytokines or transcription factors, while 13 others have as yet uncharacterized activities. The magnitude of c‐fos induction by TNF‐α inversely correlated with cell‐type‐specific cytotoxicity. Rapid and transient mRNA responses were observed in the TNF‐α‐resistant cells, whereas a slower and more persistent response was characteristic for TNF‐α‐sensitive cells. The prolonged induction of immediate early mRNAs may contribute to TNF‐α‐induced cellular cytotoxic responses.
Anti‐angiogenic activity of arachidonic acid metabolism inhibitors in angiogenesis model systems involving human microvascular endothelial cells and neovascularization in mice
Authors: Ito Ken‐Ichi , Ono Mayumi , Abe Tatsuya , Kuwano Michihiko , Morimoto Akio , Kohno Kimitoshi , Mori Teruaki , Tomita Mika , Sugenoya Akira , Nishihira Tetsuro
Published: February 2007

We have established an in vitro angiogenesis model using human omental microvascular endothelial (HOME) cells, in which epidermal growth factor (EGF) or transforming growth factor‐alpha (TGF‐α) stimulated cell migration and tube formation. In this study, we examined whether α‐guaiaconic acid (GR‐12) and its synthetic 20 derivatives showed inhibition of cell migration and tubular formation of HOME cells. We found that GR‐12 inhibits arachidonic acid metabolism, while GR‐12 and one derivative, GS‐01, inhibit tubular formation of endothelial cells in our model system. Confluent monolayers of HOME cells were damaged with a razor blade and incubated with or without TGF‐α; HOME cell migration was stimulated about 1.5‐fold over control values in the presence of TGF‐α. Treatment of HOME cells with GR‐12 or GS‐01 inhibited both spontaneous and TGF‐α‐stimulated migration. GR‐12 or GS‐01 inhibited TGF‐α‐induced HOME‐cell tube formation in type‐1 collagen gels. We examined whether these compounds could modulate tubular formation of HOME cells induced by human cancer cells. Enhanced tube formation of HOME cells by co‐cultured esophageal cancer cells was almost completely inhibited by co‐administration of GR‐12 or GS‐01. Both compounds also inhibited formation of tubular networks of HOME cells on Matrigels. We also examined anti‐angiogenic activity of these compounds in an in vivo model system of tumor angiogenesis in mice. In this system, GS‐01 inhibited development of capillary networks at a rate comparable to that of a well‐known anti‐angiogenic compound, fumagillin, but GR‐12 did not. The inhibitor of arachidonic acid metabolism is thus expected to modulate tumor angiogenesis.
Parvovirus H‐1 inhibits growth of short‐term tumor‐derived but not normal mammary tissue cultures
Authors: Van Pachterbeke C. , Tuynder M. , Rommelaere J. , Cosyn J. P. , Lespagnard L. , Larsimont D.
Published: February 2007

Infection with parvovirus H‐1 strongly interfered with the proliferation of non‐established tissue cultures derived from human breast tumors, but had little effect on the growth of corresponding normal human mammary cells. Even though tumor cells were always more sensitive to the virus than normal tissue from the same patient, appreciable quantitative differences were observed among tumor specimens. With time and sub‐cultures, the killing effect of the virus on tumor cells became amplified. The impaired growth of infected tumor cells was due both to cytotoxic and to cytostatic action of H‐1 virus and was associated with their greater capacity for virus‐DNA amplification as compared with normal cells.
Increased accumulation of p53 protein in cisplatin‐resistant ovarian cell lines
Authors: Brown Robert , Clugston Carol , Edlin Angela , Vasey Paul , Kaye Stanley B. , Burns Philip , Vojtěšek Borek
Published: February 2007

We have examined p53 protein levels in cell lines selected for resistance to the chemotherapeutic drug cis‐diamminedichloroplatinum (II), cisplatin. The majority of the independent cisplatin‐resistant clones isolated by a single selection with cisplatin from the ovarian tumour cell line A2780 showed increased levels of p53 protein compared to the parental cell line. Elevated p53 protein levels were also observed in cisplatin‐resistant ovarian human tumour lines isolated after multiple exposures to cisplatin (A2780/cp70 and OVIP/DDP). Direct PCR sequencing of p53 cDNAs showed that both the A2780/cp70 and the parental A2780 cell lines had a wild‐type p53 gene sequence. The OVI P and OVI P/DDP lines both had a heterozygous mutation at codon 126. Cell‐cycle analysis after gamma‐irradiation or cisplatin treatment showed evidence of a G1/S and G2/M cell‐cycle checkpoint in both A2780/cp70 and the sensitive parental cell lines. However, the resistant cell line A2780/cp70 showed less inhibition of DNA synthesis after gamma‐irradiation than the sensitive cell line. Transfection of a mutant p53 gene construct (containing a mutation at codon 143, val to ala) into the A2780/cp70 resistant cells conferred a significantly increased sensitivity to cisplatin, suggesting that p53 is a direct determinant of cisplatin resistance in these cells. However, expression of this mutant p53 in the A2780 cells did not affect sensitivity.
Mitotic activity, apoptosis and TRPM‐2 mRNA expression in dmba‐induced rat mammary carcinoma treated with anti‐estrogen toremifene
Authors: Huovinen Riikka , Wärri Anni , Collan Yrjö
Published: February 2007

Anti‐estrogen toremifene inhibits growth of the 7,12‐dimethylbenz(a)anthracene(DMBA)‐induced rat mammary carcinoma. The changes in proliferation and cell death were studied in detail. Proliferation was measured by counting mitotic figures in histologic sections, expressed by volume‐corrected mitotic index (M/V INDEX). Respective volume corrected apoptotic index (A/V INDEX) was measured by counting apoptotic nuclei in the same sections. The presence of apoptotic cells was confirmed by transmission electron microscopy. In the untreated tumors, both mitosis and apoptosis were frequent. In the toremifene‐treated tumors, the mean M/V INDEX was about half of the mean M/V INDEX in the untreated control tumors. The mean A/V INDEX in the toremifene‐treated tumors was about % of the mean A/V INDEX in the controls. In toremifene‐treated tumors, A/V INDEX was strongly correlated with TRPM‐2‐gene expression, which was also enhanced when compared with the controls. TRPM‐2 gene has been associated with programmed cell death induced by hormonal ablation in prostate‐ and breast‐cancer cells. No such correlation was seen in control tumors. These findings suggest that, in the DMBA‐tumor model, toremifene affects the cell turnover by inhibiting mitotic activity and modifying abundant spontaneous apoptosis. In this model, the inhibition of tumor growth results mostly from reduction of mitotic activity.
Nerve growth factor effects on human and mouse melanoma cell invasion and heparanase production
Authors: Marchetti Dario , Menter Dave , Jin Li , Nicolson Garth L. , Nakajima Motowo
Published: February 2007

The role of growth factor networks in regulating the progression of human melanocytes towards tumorigenicity and ultimately the malignant phenotype is poorly understood. In particular, the autocrine and paracrine influences that modulate cellular invasion and extracellular matrix degradative enzymes of melanoma cells remain undefined at the molecular level. We report here that nerve growth factor (NGF) can modify some metastasis‐associated cellular properties of human and mouse melanoma cells. Treatment of early‐passage human metastatic melanoma cells (MeWo) or their variants (3S5, 70W) with biologically active 2.5S NGF resulted in (a) delayed density‐dependent inhibition of melanoma cell growth; (b) increased in vitro invasion through a reconstituted basement membrane; and (c) time‐ and dose‐dependent induction of heparanase, a heparan‐sulfate‐specific endo‐β‐D‐glucuronidase associated with human melanoma metastasis. These effects of NGF were most marked in the 70W brain‐colonizing cells (70W > MeWo > 3S5). The NGF enhancement of heparanase secretion was not species‐specific, since it was also observed in murine B16 melanoma cells; the highest NGF stimulation of heparanase was found in brain‐colonizing murine B16‐B15b variant (B16‐B15b > B16‐BL6, B16‐F10, B16‐F1). NGF also increased the invasive capacity of the human 70W and murine B16‐B15b sublines in a chemoinvasion assay performed with filters coated with purified heparan sulfate proteoglycan (HSPG). The enhancement of chemotactic response and heparanase production was detected at NGF concentrations sufficient to fully saturate both low‐ and high‐affinity NGF receptors (NGFR), the neurotrophin receptor (p75) and the trkA gene product, respectively. The results suggest that, in addition to the effects of NGF on cellular development and differentiation within the peripheral and central nervous systems, NGF can exert changes in the invasive properties of neuroectoderm‐derived melanoma cells.
Working group report on hepatitis viruses
Authors: Vainio H. , Heseltine E. , Møller H.
Published: February 2007

Viruses play a major role in the etiology of a number of malignancies, including hepatocellular carcinoma (HCC) and cervical cancer, which are common in many parts of the world. In 1991, an ad hoc advisory group was convened by the International Agency for Research on Cancer (IARC) to consider whether viruses and other biological agents should be included in the IARC Programme for the preparation of Monographs on the Evaluation of Carcinogenic Risks to Humans. The group agreed unanimously that the carcinogenic risks of biological agents should be evaluated by expert groups such as those that have been convened by IARC over the past 20 years to discuss chemicals, physical agents, industries and cultural habits. A list of biological agents to be evaluated was drawn up, and the priority of each was designated by consensus on the basis of considerations regarding both their importance to public health and the status of the available epidemiological and biological evidence.

On 8‐15 June 1993, IARC convened a Working Group consisting of experts in epidemiology, experimental carcinogenicity, internal medicine, pathology, communicable diseases and molecular biology (see list of participants) to discuss 3 viruses given high priority by the ad hoc advisory group, i.e., hepatitis B virus (HBV), hepatitis C virus (HBC) and hepatitis D virus (HDV, or the delta agent).

These 3 viruses all cause a similar acute illness involving the liver, but they are very different in structure and biology. Their common feature is that the liver disease they cause may have a chronic course; they differ in that way from hepatitis A and hepatitis E viruses, for which no association with chronic liver infection has been recognized. Furthermore, no indication is available to suggest that hepatitis A virus leads to HCC or any other cancer, and no studies have been reported on hepatitis E virus. The considerations of the Working Group were thus limited to HBV, HCV and HDV.
Characterization of a monoclonal antibody against human prolactin receptors
Authors: Banerjee Rina , Ginsburg Erika , Vonderhaar Barbara K.
Published: February 2007

Tamoxifen is the first line of therapy for most human breast cancers. It not only works through the estrogen receptor but also can directly affect the binding of prolactin to its receptor. To define this latter mechanism, the nature of the prolactin receptor needs to be clearly defined. Monoclonal antibody (MAb) B6.2, an IgG1 raised against a membrane‐enriched fraction from metastatic human breast cancer cells, was as effective as polyclonal anti‐prolactin receptor antibody in inhibiting the binding of prolactin to membranes from human tissue and to T47D human breast cancer cells. Control MAbs, MOPC‐21 and the anti‐NCA B1.1 MAb, had no effect on binding. Epidermal growth‐factor receptors on these same cells were unaffected by B6.2. Prolactin‐induced growth of the T47D cells was blocked by addition of B6.2 to the media while the control antibodies were without effect. Specific binding of B6.2 to the cells was completely inhibited by prolactin. Binding of both prolactin and B6.2 was inhibited by growing the T47D cells in the presence of tunicamycin A, under conditions where protein synthesis was not affected but glycosylation of proteins was. An affinity column of B6.2 was used to purify its antigen from T47D cells. The primary purification product, a Mr 90,000 protein, specifically bound the lactogenic hormones human prolactin, human growth hormone and ovine prolactin but not the somatogenic hormone, bovine growth hormone and was precipitated by the polyclonal anti‐prolactin receptor antibody but not by control MAbs. When tryptic and V8 digests of the B6.2 antigen and purified prolactin receptors were compared, identical electrophoretic profiles were obtained. Mouse 3T3 cells, when stably transfected with the gene for the long form of the human prolactin receptor, reacted with B6.2 and polyclonal anti‐prolactin receptor antibody. Parental 3T3 cells, devoid of prolactin receptors, were negative for all antibodies tested. Thus, MAb B6.2 provides a useful tool for further studies on purification and characterization of these receptors from human tissues and may provide new insights into treatment for breast cancer.
Non‐viral risk factors for nasopharyngeal carcinoma in the philippines: Results from a case‐control study
Authors: West Sheila , Hildesheim Allan , Dosemeci Mustafa
Published: February 2007

In a case‐control study of NPC conducted in the Philippines, 104 predominantly non‐Chinese (<10% ethnically Chinese) cases of nasopharyngeal carcinoma (NPC) and 205 hospital and community controls were recruited. Risk factor information was obtained through personal interview. The occupational history of each subject was reviewed “blind” by an industrial hygienist to determine estimates of exposure to formaldehyde, solvents, dusts, exhaust and pesticides. After control for confounding, subjects who were first exposed to formaldehyde 25 or more years prior to diagnosis/interview or who were first exposed before the age of 25 were found, in relation to those never exposed, to be at a 4.0‐fold excess risk of disease. Similarly, those first exposed to dust and/or exhaust 35 or more years prior to diagnosis/ interview were at a 4.4‐fold excess risk of disease and those first exposed before the age of 20 were at a 3.5‐fold excess risk of disease. Salted fish consumption was not associated with risk, while consumption of processed meats protected against NPC. Smoking was positively associated with NPC, but only when cases were compared to community controls. Relative to non‐smokers, subjects reporting more than 30 years of smoking were at an adjusted 7.2‐fold excess risk of disease. Herbal medicine use and burning of anti‐mosquito coils were both independently associated with risk of NPC, with ever‐users of herbal medicines being at a 2.5‐fold excess risk of disease and those reporting daily use of anti‐mosquito coils being at a 5.9‐fold excess risk of disease relative to never users. Exposure to solvents, pesticides, or use of betel nuts were not associated with NPC risk.
Chemotherapy‐induced differential changes in lymphocyte subsets and natural‐killer‐cell function in patients with advanced breast cancer
Authors: Sewell Herb F. , Halbert Cyril F. , Robins R. Adrian , Galvin Alison , Chan Stephen , Blamey Roger W.
Published: February 2007

To elaborate a rational approach to chemoimmunotherapy in humans, information is required as to how current cytotoxic chemotherapy regimens modulate patients' endogenous immune cells. We have studied a group of 16 advanced breast cancer patients who received cyclical cytotoxic chemotherapy (CMF‐cyclophosphamide, methotrexate and 5‐fluorouracil) and have documented the progressive differential effects of chemotherapy on endogenous immune cells as judged by changes in immunophenotype and absolute numbers of lymphocyte subsets, together with analysis of natural‐killer‐cell function. Cells with the immunophenotype of natural killer cells and lympho‐kine‐activated killer cells (NK/LAK cells) were well retained, but their function was suboptimal. Additionally, CD8 T cells were well preserved, but the numbers of CD4 T cells decreased with succeeding cycles of chemotherapy; B‐cell numbers decreased rapidly from the first cycle of chemotherapy. These cellular changes in humans indicate defined and precisely timed windows of opportunity for introducing in vivo, simple and direct immune stimulation of the cells modulated by chemotherapy, with the possibility of improving therapy and survival in this disease.
Detection of circulating intercellular adhesion molecule‐1 in hepatocellular carcinoma
Authors: Hyodo Ichinosuke , Jinno Kenji , Tanimizu Masahito , Hosokawa Yasushi , Nishikawa Yoshiyuki , Akiyama Masanori , Mandai Koichi , Moriwaki Shousuke
Published: February 2007

Serum levels of circulating intercellular adhesion molecule‐1 (cICAM‐1) were measured in 23 patients with chronic hepatitis (CH), 22 with liver cirrhosis (LC) and 45 with hepatocellular carcinoma (HCC) using an ELISA. Serum samples from all patients showed significantly higher cICAM‐1 levels than serum from 50 normal controls. The cICAM‐l level was significantly increased in LC or HCC when compared with CH, but no differences were noted between LC and HCC. Levels of cICAM‐1 correlated well with serum bilirubin, retention rate of indocyanine green, hyaluronic acid, type IV collagen 7‐S and type III procollagen peptide levels but not with tumor size or circulating tumor markers (α‐fetoprotein and des‐γ‐carboxyprothrombin). Our findings indicate that the measurement of cICAM‐l is useful for the determination of the severity of liver disease and hepatic fibrosis. HCC tissues obtained from 10 patients were immunohistochemically stained for ICAM‐I. Enhanced ICAM‐I expression was found on the tumor cell membranes. Sequential measurements of cICAM‐1 levels showed that they changed in a similar manner to those of a‐fetoprotein during the course of treatment of HCC in a patient with very high pretreatment levels of both markers. These results suggest that HCC cells shed ICAM‐1 into the circulation. We conclude that cICAM‐1 is not a diagnostic marker for HCC, but may be useful for monitoring the response to treatment.
Screening of human bladder tumors and urine sediments for the presence of H‐ras mutations
Authors: Levesque Peter , Ramchurren Nirasha , Saini Kulvinder , Joyce Adrian , Libertino John , Summerhayes Ian C.
Published: February 2007

A series of 111 human bladder tumors were screened using oligonucleotide mutant specific probes, restriction enzyme analysis and single‐stranded confirmation polymorphism (SSCP) for the presence of H‐ras activation events. Thirty‐three tumors were found to harbor H‐ras mutations where a glycine to valine (G→ T) change in codon 12 was the most common point mutation recorded (26 tumors). Additional mutations involved glycine to cysteine at codon 13 (2 tumors) and glutamine to arginine/lysine/leucine at codon 61 (3/1/1 tumors, respectively). Ambiguous signals recorded with oligonucleotide probes were further analyzed using SSCP analysis revealing the presence of H‐ras mutations in restricted regions of some tumors. The apparent sensitivity of SSCP enabled us to extend this study to DNA isolated from urine sediments where 4 of the 9 patients studied showed representation of mutant H‐ras. Our study demonstrates a sensitive, non‐invasive assay for the screening of urine‐borne cells, with no requirement for prior knowledge of the mutational change at the H‐ras locus.
Characterization of cell lines established from merkel‐cell (“small‐cell”) carcinoma of the skin
Authors: Leonard J. Helen , Bell John R. , Kearsley John H.
Published: February 2007

Six cell lines have been established from different biopsies of the rare small‐cell skin cancer of neuro‐endocrine origin known as Merkel‐cell carcinoma (MCC). These were established from metastatic lesions and have now been in culture for periods varying from 1 to 3 years. All lines grow as suspension cultures and exhibit typical MCC cytological features of small round cells with little cytoplasm, round nuclei and tight junctions. There was variation in the number and size of nucleoli and dense‐core granules. We present details of their immunohistochemistry and growth characteristics in culture. The latter were similar to small‐cell lung‐cancer (SCLC) lines and can be subdivided, by their appearance, into type I, type II and type III in the same way as SCLC lines. This classification may be of significance for the prognosis and management of MCC.
A CD16/CD30 bispecific monoclonal antibody induces lysis of hodgkin's cells by unstimulated natural killer cells In AND In vivo
Authors: Hombach Andreas , Jung Wolfram , Pohl Christoph , Renner Christoph , Sahin Ugur , Schmits Rudolf , Wolf JÜRgen , Kapp Ursula , Diehl Volker , Pfreundschuh Michael
Published: February 2007

In order to target NK cells against the Hodgkin's‐derived cell line L540, we developed bispecific monoclonal antibodies (Bi‐MAbs) by somatic hybridization of the 2 mouse hybridoma cell lines HRS‐3 and A9 which produce monoclonal antibodies (MAbs) with reactivity against the Hodgkin and Reed‐Sternberg cell‐associated CD30 antigen and the CD 16 antigen (FcγIII receptor), respectively. The CD 16 MAb‐producing cell line A9 was selected as a partner for HRS‐3 because of its efficiency in inducing lysis of the A9 hybridoma cells by resting NK cells. The hybrid hybridoma cell line HRS‐3/A9 produced the supernatant with the strongest bispecific reactivity and was repeatedly subcloned and used for ascites production. Crude supernatant and purified HRS‐3/A9 Bi‐MAb triggered specific lysis of the CD30+ Hodgkin's‐derived cell line L540, but not of the CD30− cell line HPB‐ALL by unstimulated peripheral‐blood lymphocytes and NK‐cell‐enriched populations. Moreover, treatment of SCID mice bearing heterotransplanted human Hodgkin's tumors with HRS‐3/A9 and human peripheral blood lymphocytes induced specific complete tumor regression in 10/10 animals. We thus report successful tumor treatment in an in vivo model using NK‐cell‐associated Bi‐MAbs and show that the Bi‐MAb HRS‐3/A9 is an efficient promoter of the anti‐tumor effects of NK cells in vitro and in vivo.
Quantitative and/or qualitative changes in the P21‐H‐ras post‐translational products in regenerating liver and during hepatocarcinogenesis
Authors: Gulbis Beatrice , Alexandre Kathy , Galand Paul
Published: February 2007

Using Western immunoblot analysis with the Ras‐10 monoclonal antibody (MAb), we characterized 4 post‐translational products of the c‐H‐ras gene in rat tissues, and showed that they were readily distinguished from the normal p2l‐N‐ras or Val12 mutant p21‐ras products. In the present study, we used this approach to compare the electrophoretic pattern of p21 ‐H‐ras during chemically induced hepatocarcinogenesis in rats with that in control liver samples. Three types of pattern were defined in the liver samples taken at various stages of malignant progression. The first type, like all the 18 normal samples analyzed, was characterized by a predominant intensity of spot a (corresponding to the palmitoylated p21 ‐H‐ras product). This was observed in all the samples at the stage of foci and only in a proportion of nodular or tumoral tissues. The second type of pattern deviates from this normal basal pattern by a higher relative level in one or more of the precursors of the fully processed p21‐H‐ras product. It was observed in all liver samples at the stage of nodules and in a proportion of tumors, but also in all samples from fetal or regenerative liver, thus suggesting an association with high proliferative activity of the hepatocytes. The third type of pattern was characterized by the presence of spots never detected in any of the normal rat tissue or cells that we investigated. Abnormal spots were observed in nodular liver samples and in hepatocarcinomas, indicating that they probably correspond to mutant p21 ‐H‐ras products. The fact that the 2 types of abnormal products were not constantly associated with neoplasms indicates that if they play a role in their induction or maintenance this may also be achieved in an independent way, perhaps, but not necessarily, involving another mutation of the ras gene.
α galnac is essential for recognition of EXO‐1 epithelial antigen by mouse monoclonal antibody Pa‐G‐14
Authors: Schröder Helmut , Francí Clara , Egea Gustavo , Oriol Rafael , Klingel Reinhard , Dippold Wolfgang G. , Real Francisco X.
Published: February 2007

Mouse monoclonal antibody Pa‐G‐14 detects Exo‐1, an antigen whose expression is regulated in the processes of epithelial‐cell differentiation and transformation. The epitope recognized by Pa‐G‐14 is present both in glycosphingolipids and in mucin glycoproteins. To characterize the specificity of Pa‐G‐14, immuno‐thin‐layer chromatography, biochemical, and enzymatic treatment of glycosphingolipid extracts from human pancreas were used. The antibody bound to all blood‐group‐A substances; αGaINAc, but not fucose, was essential for reactivity. In ELISA, Pa‐G‐14 also reacted with ovine and bovine submaxillary mucins but not with porcine submaxillary mucin. Binding to ovine submaxillary mucin was resistant to neuraminidase treatment. In solid‐phase absorption assays on synthetic carbohydrate structures, Pa‐G‐14 recognized broadly blood group A, Tn and sialyl‐Tn. Using immuno‐electron‐microscopic techniques, reactivity with all Golgi cisternae and mucin droplets of mucous cells in ovine submaxillary gland was demonstrated. All these assays indicate that Pa‐G‐14 shows a novel specificity, since it binds blood group A, Tn and sialyl‐Tn, the common structural feature of these epitopes being the presence of a terminal αGalNAc sugar unit.
Immunohistochemical survey of pS2 expression in intraductal lesions associated with invasive ductal carcinoma of the breast
Authors: Inaji Hideo , Koyama Hiroki , Motomura Kazuyoshi , Noguchi Shinzaburo , Tsuji Naoko , Wada Akira
Published: February 2007

We examined the expression of pS2 protein in 48 invasive ductal breast carcinomas with an extensive intraductal component, using immunohistochemical staining of paraffin‐embedded sections. The patients selected for this study would have met the criteria for breast‐conserving surgery applied at our institute at present. The rate of pS2 expression in the intraductal lesion was significantly higher than that in the main invasive lesion. The incidence of pS2 protein expression in the latter lesions was very similar to that in invasive carcinoma without intraductal lesions. The pS2 positivity of the intraductal lesion was equal to or higher than that of the invasive lesion. Of intraductal lesions, those classified as non‐comedo carcinomas frequently contained more pS2 protein than did comedo carcinomas.
Low incidence of C‐Ha‐ras gene mutations in benign and malignant cutaneous lesions from transplant recipients
Authors: Pélisson I. , Chardonnet Y. , Euvrard S. , Schmitt D.
Published: February 2007

Transplant recipients successively develop benign, pre‐malignant and malignant skin lesions on sun‐exposed areas. It has been suggested that UV radiations might induce mutations in ras oncogenes and p53 tumour‐suppressor gene, responsible for skin cancers. With PCR and oligoprobe hybridization, we investigated c‐Ha‐ras gene mutations at codons 12 and 61 in 120 cutaneous lesions from grafted patients, since they could represent a marker of the evolution of benign skin lesions towards malignancy in this population; 29 similar skin biopsies from non‐immunosuppressed patients were also analyzed. In transplant recipients, we detected mutations at codon 12 only in 1/42 non‐melanoma skin cancers and 2/29 pre‐cancerous keratoses. No mutation was detected in 11 cases of cutaneous Bowen's disease from grafted patients and in pre‐malignant and malignant skin samples from control patients. Benign warts exhibited an overall incidence of 18% and 15% of mutations at codon 12 of c‐Ha‐ras gene in grafted and control patients respectively. We detected only one mutation at codon 61 in a plantar wart. Human papillomaviruses (HPV) are thought to be involved in the malignant evolution of cutaneous disorders in transplant recipients and cooperate with a ras oncogene to induce malignancy in vitro. The presence of HPV ONA in our series of skin samples from grafted patients showed no correlation with the occurrence of c‐Ha‐ras mutations. Our findings indicate that c‐Ha‐ras‐gene activation by mutations is rare in cutaneous lesions from transplant recipients, and is unlikely to play a crucial role in transformation towards malignancy in skin carcinogenesis among grafted patients.
Expression and shedding of ICAM‐1 in bladder cancer and its immunotherapy
Authors: Jackson Andrew M. , Alexandrov Anton B. , Gribben Shona C. , Esuvarnathan K. , James Keith
Published: February 2007

Intercellular adhesion molecule‐1 (ICAM‐1) is one of 3 major ligands for the β2 integrin leucocyte function‐associated antigen‐ I (LFA‐1). Several reports have emerged describing soluble forms of ICAM‐1 in association with normal and pathological states (e.g., malignancy). In this study we have identified the secretion of soluble ICAM‐1 in tissue culture supernatants from bladder tumour monolayers and in the urine of patients receiving intravesical BCG immunotherapy for superficial bladder cancer. In vitro, small amounts of sICAM‐1 were detected in the tissue culture supernatants of bladder cancer cells, known to constitutively express ICAM‐1. Following stimulation with interferon γ, the levels of sICAM‐1 increased inversely to the levels of cell surface ICAM‐1, suggesting shedding. Induction and augmentation of cell surface ICAM‐1 required de novo mRNA and protein synthesis. However, treatment with cycloheximide, after stimulation with IFN‐γ, resulted in increased levels of membrane associated ICAM‐1. Correspondingly, the level of sICAM‐1 in the supernatant was low in comparison with controls, suggesting that cycloheximide acted via stabilization of membrane ICAM‐1 or via prevention of some enzymatic cleavage event. In vivo, sICAM‐1 can be detected at high levels in patients' urine following immunotherapy of bladder cancer with intravesically administered BCG organisms. Production of sICAM‐1 is transient and occurs only in the first 12 hr following instillation. Furthermore, production of sICAM‐1 is heterogeneous as some patients fail to produce any at all. If the source of sICAM‐1 is the bladder tumour per se, then its detection in urine could indicate a response of the tumour to immunotherapy and indeed may prove a useful indicator of clinical response.
Normal breast epithelial cells produce interleukins 6 and 8 together with tumor‐necrosis factor: Defective il6 expression in mammary carcinoma
Authors: Basolo Fulvio , Conaldi Pier Giulio , Fiore Lisa , Calvo Simonetta , Toniolo Antonio
Published: February 2007

Virtually pure primary cultures of normal mammary epithelial cells (MEC) obtained from healthy women were shown to release interleukin 6 and 8 (IL6, IL8) and to produce a non‐secreted form of tumor‐necrosis factor (TNF). No interferon (IFN), whether α, β or γ, or ILI‐α or ‐β could be detected. Analysis of cellular RNA confirmed these findings and showed that MEC also express IL6 receptor and TNF‐α‐related mRNAs. Epithelial cells were selectively stained by antibodies to IL6, IL8 and TNF‐α both in primary cultures and in the normal mammary gland. Samples of human milk contained sizable amounts of IL6, IL8 and IFN‐γ; yet the liquid phase was consistently negative for other cytokines (i.e., TNF‐α, IFN‐α/‐β, ILI‐α/‐β). Expression of IL6 (but not of IL8 and TNF‐α) was abolished in ductal infiltrating carcinomas and greatly reduced in cultures of oncogene‐transfected mammary cells, suggesting that alterations of IL6 expression are associated with pathogenesis in breast cancer.
Specificity and properties of MAb RS7‐3G11 and the antigen defined by this pancarcinoma monoclonal antibody
Authors: Stein Rhona , Basu Amartya , Chen Susan , Shih Lisa B. , Goldenberg David M.
Published: February 2007

The murine monoclonal antibody (MAb) RS7‐3G11 is an IgG1 with pancarcinoma reactivity, which has been raised against human squamous‐cell carcinoma of the lung. Immunoperoxi‐dase staining of frozen tissue sections demonstrated that the antigen defined by RS7‐3G11 is present in tumors of the lung, stomach, bladder, breast, ovary, uterus and prostate. The rate and extent of internalization of RS7‐3G11 into Calu‐3, an adenocarcinoma of the lung cell line, was investigated using unconjugated MAb, followed by fluorescence labeling, and by binding 125l‐RS7‐3G 11 followed by acid removal of surface‐bound antibody. Rapid internalization of MAb RS7‐3G11 into target cells was observed. Antibody internalization was noted at 30 min, and by 2 hr virtually all MAb RS7‐3GII was internal. Although MAb RS7‐3G11 was raised against non‐small‐cell carcinoma of the lung, ME‐180, a cervical‐carcinoma cell line, expresses higher quantities of the antigen than the lung carcinoma cell lines. Due to the higher antigen density in ME‐180 cells, this line was used for immunoprecipitation studies and antigen purification. Immunoprecipitation studies using the ME‐180 cervical‐carcinoma cell line metabolically labeled with [3H] leucine or [3H]glucosamine demonstrated that the antigen defined by RS7‐3G11 is a glycoprotein of Mr 46 kDa. Deglycosylation by treatment with endoglycosidase‐F resulted in a protein with a Mr of 35 kDa. RS7‐3G11 ‐antigen was purified from ME‐180 tissue‐culture cells using affinity‐column chromatography. By SDS‐PAGE it was seen that the antigen was highly purified. The major band appeared at Mr of 45 to 48 kDa. This result is in agreement with the immunoprecipitation studies. The broad band observed in the SDS‐PAGE is typical of many glycoproteins, and suggests heterogeneity of glycosylation. Chemical and enzymatic treatments of the antigen, followed by Western blot analyses, suggest that the RS7‐3G11 antigenic determinant is composed of a conformation‐dependent peptide.
Alterations of the p53 tumor‐suppressor gene in transformed mouse liver cells
Authors: Sun Yi , Hegamyer Glenn , Nakamura Kazuki , Kim Hyungtae , Oberley Larry W. , Colburn Nancy H.
Published: February 2007

Mutational inactivation of p53, a potential tumor‐suppressor gene, has been found in many tumors of humans as well as rodents. The p53 status in normal and transformed mouse liver cell lines has, however, not been investigated. We examined possible point mutations and compared mRNA and protein expression of the p53 gene in normal vs. transformed mouse liver cells. The transformed cells studied included lines spontaneously transformed by sub‐culture, virally transformed by simian virus 40 (SV40), and chemically transformed by N‐methyl‐N‐nitro‐N‐nitrosoguanidine (MNNG) or methylcholanthrene epoxide (MC). A heterozygous G → A point mutation at codon 241, position I, of p53 was detected in MNNG‐transformed cells after screening of 5 evolutionarily conserved regions where mutation hot‐spots are clustered. The mutation causes a glyarg substitution. No mutations were found in normal or other transformed cells. The steady‐state levels of p53 mRNA were decreased in chemically transformed (both MNNG‐ and MC‐transformed) cells. Elevated levels of p53 protein were found in spontaneously transformed and SV40‐transformed cells, an observation that may reflect a longer half‐life of the protein, as has been shown in other transformed lines. The low level of the p53 protein in MC‐transformed cells may result from transcriptional depression of the p53 gene. We conclude from these data that abnormal p53 status, such as point mutation or altered expression, may play a role during the malignant transformation of mouse liver cells.
Effect of serum and cell density on transmembrane distribution of cAMP and cGMP in transformed (C4‐i) and non‐transformed (WI‐38) human cells
Authors: Ørbo A. , Jaeger R. , Sager G.
Published: February 2007

The ratio between cGMP and cAMP in plasma/urine is elevated in several types of malignancies. The present in vitro study showed that the ratio between extracellular cGMP and cAMP increased during the proliferation of C4‐1 cells (derived from a carcinoma of the uterine cervix), whereas this ratio decreased in W1‐38 cells (normal lung fibroblasts). These results can be explained by differences between the transformed and non‐transformed cells in the cell‐density‐dependent transmembrane distribution and intracellular levels of cyclic nucleotides. In the serum‐deprived cultures, no profound effects were seen on the cell‐density‐dependent biokinetics of cAMP and cGMP. In the absence of serum, growth of C4‐1 cells was markedly retarded, whereas W1‐38 cells were unable to expand at all.
Effect of epidermal growth factor and transforming growth factor β1 on growth and invasive potentials of newly established rat bladder carcinoma cell lines
Authors: Kawamata Hitoshi , Kameyama Shuji , Nan Li , Kawai Koji , Oyasu Ryoichi
Published: February 2007

We established 5 rat bladder cell lines (MYU3L, MYU4, MYU6s, MYKUIL and MYP3). EGF stimulated DNA synthesis of all the cells in monolayer culture, regardless of the number of EGF receptors. In soft agar, only MYU3L formed colonies, and EGF enhanced their growth. However, EGF did not induce the other cells to grow in soft agar. In contrast, TGF‐β1 inhibited the growth of the cells, but a tumorigenic cell and the cells which were established from large in vivo tumors were more resistant than the others to TGF‐β1. We tested the effect of growth factors on the invasive potential of MYP3 cells (non‐tumorigenic), MYU3L cells (tumorigenic/highly invasive but not metastatic) from newly established cell lines, and another metastatic cell line, LMC 19. MYP3 expressed only a trace amount of 92‐kDa gelatinase (MMP‐9), whereas MYU3L expressed interstitial collagenase (MMP‐1) and MMP‐9, and LMC19 expressed 72‐kDa gelatinase (MMP‐2) and MMP‐9. The release of MMP‐2 in LMC 19 was stimulated by TGF‐β1, but EGF had no effect on the release of any MMPs in either type of cells. These observations suggest that EGF acted as a mitogen on all the cells tested, but did not enhance the malignant phenotype. Further, the loss of responsiveness to the suppressive effect of TGF‐β1 may be an important step toward a malignant phenotype. Some of malignant tumors may utilize TGF‐β1 for enhancing their invasive and metastatic potential.