bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple mechanisms of resistance in a series of human testicular teratoma cell lines selected for increasing resistance to etoposide
Authors: Hosking Louise K. , Whelan Richard D. H. , Shellard Sharon A. , Davies Sally L. , Hickson Ian D. , Danks Mary K. , Hill Bridget T.
Published: February 2007

Mechanisms of resistance to VP‐16 were monitored in a series of sublines of the human testicular teratoma cell line (SuSa) derived following exposure either to fractionated X‐irradiation (DXR‐10) or to VP‐16 using pulsed 24‐hr exposures (VP 10) or continuous exposure conditions (VPC2, VPC3 and VPC4). Orders of resistance expressed (ranging from 3‐ to 33‐fold based on ICS0 values derived from colony forming assays) were comparable with those likely to be encountered clinically, All of these resistant sublines showed some cross ‐resistance to VCR, and the 3 drug‐selected sublines tested also proved cross‐resistant to ADR. Resistance was not associated with modified 3H‐VP‐16 accumulation. However, decreased VP‐16‐induced SSBs were detectable in all the resistant sublines and a strong positive correlation was noted between the extent of SSB formation and VP‐16 resistance by linear regression analysis. Topo IIα protein content, as judged by Western blotting, was significantly decreased only in the sublines derived by continuous exposure to VP‐16, but this was not progressive with increasing levels of resistance expressed. RNase protection assays also showed no significant differences in Topo IIα expression in the low‐level resistant DXR‐10 and VP 10 sublines, contrasting with the 2‐fold decreases identified in the VPC2, VPC3 and VPC4 sublines. Significantly, however, mRNA levels of two alternately spliced Topo IIβ mRNAs were markedly decreased (2‐ to 9‐fold) in all the drug‐selected resistant sublincs. No mutations in consensus ATP‐binding sequences or in the DNA‐binding region of Topo Ma were detected by single strand conformations I polymorphism analysis. Significant Pgp over express ion was only identified in the most highly resistant sublines VPC3 and VPC4, which both showed 4‐fold cross‐resistance to VCR. Decreased 3H‐VCR accumulation and partial reversal of resistance by VPM (6.6 μM) addition was also identified, consistent with a functional Pgp being overexpressed in these sublines. Modifications of Topo II expression therefore appear to precede Pgp overexpression in this series of sequentially derived VP‐16 resistant sublines and to represent the predominate mechanism underlying low level (< 10‐fold) resistance. © 1994 Wiley‐Liss, Inc.
Cytoreductive effects of anti‐transferrin receptor immunotoxins in a multicellular tumor spheroid model
Authors: Chignola Roberto , Foroni Roberto , Candiani Carola , Franceschi Antonia , Pasti Murcella , Stevanoni Gabriella , Anselmi Cristina , Tridente Giuseppe , Colombatti Marco
Published: February 2007

We have evaluated the sensitivity to immunotoxins (IT) of monolayer and of 200‐250 μM multicellular tumor spheroid (MTS) cultures obtained with human breast (MCF7) and glioblastoma (U 118) tumor cells and with rat glioblastoma (9L) cells. Monolayer MCF7 and U118 cells were highly sensitive to anti‐transferrin receptor (anti‐TfnR) ricin A chain (RTA)‐IT (Tfn‐RTA and Mab OKT9‐RTA) treatment in the presence of the intracellular RTA‐IT enhancing agent human serum albumin monensin (HSA‐Mo) conjugate. A 790‐ to 2000‐fold higher concentration of anti‐TfnR IT was instead required to reduce by 50% the volume of individually treated MCF7 spheroids, as evaluated by applying the Gompertz growth model. Monolayer 9L cells showed 230‐ to 5700‐fold lower sensitivity to Tfn‐RTA IT than MCF7 and UI 18 monolayers, yet 9L spheroid cells were almost as sensitive to anti‐TfnR IT as monolayer 9L cultures. Binding studies performed with [125I]‐Tfn and FITC‐labelled anti‐TfnR MAb revealed that 9L monolayers and MTS expressed 4.1‐fold and 8.8‐fold lower amounts of TfnR than MCF7 monolayers and MTS, respectively. However, Tfn bound to TfnR sites of 9L and of MCF7 cells with comparable affinity. Experiments carried out with the diphtheria toxin mutant CRMI07 linked to Tfn con‐firmed the pattern observed with RTA‐IT. Monolayers and spheroids showed no considerable differences in sensitivity to ricin toxin. Collectively, these results indicated that the efficacy of IT against 3‐D tumors is heavily influenced by the number of target Ag expressed by the tumor cells, as well as by the affinity of IT/toxin‐cell interaction. © 1994 Wiley‐Liss, Inc.
Expression of proliferating cell nuclear antigen (PC10), p53 protein and c‐erbB‐2 in renal adenocarcinoma
Authors: Lipponen Pertti , Eskelinen M. , Hietala K. , Syrjänen K.
Published: February 2007

Expressions of proliferating cell nuclear antigen (PC 10), p53 protein (CMI) and c‐erbB‐2 (NCL‐I) were immunohistochemically analysed in 123 renal adenocarcinomas with known follow‐up data. e‐erbB‐2 protein was weakly and focally expressed in 10% of the tumours, and the expression was not related to clinical or histological variables or survival. p53 protein was expressed in 33% of the tumours. Expression of p53 protein was independent of stage, grade and prognosis, while expressions of c‐erbB‐2 and p53 were weakly interrelated. Proliferating cell nuclear antigen was expressed in all tumours, and the fraction of PC 10‐positive nuclei was significantly related to grade, stage and prognosis. Multivariate analysis of clinical, histological and immunohistochemical prognostic factors indicated that the extent of the tumour, its histological differentiation and proliferation rate of the cancer cells are the most important prognostic factors. Recurrence‐free survival was related to the fraction of PCIO‐positive nuclei, histological differentiation, sex and expression of p53 protein. Over‐expression of p53 protein was related to a long recurrence‐free survival. Our results show that PC10 immunolabelling can be used to determine the prognostic category in renal adenocarcinoma, whereas the expressions of p53 protein or c‐erbB‐2 are only weak prognostic indicators. © 1994 Wiley‐Liss, Inc.
Effect of protein kinase C inhibitors on invasiveness of human melanoma clones expressing different levels of protein kinase C isoenzymes
Authors: Mapelli Elena , Banfi Patrizia , Sala Elena , Sensi Marialuisa , Supino Rosanna , Zunino Franco , Gambetta Romolo A.
Published: February 2007

The involvement of protein kinase C (PKC) in the mechanism of chemotaxis and invasiveness of human melanoma has been studied in 6 clones of 665/2 cell line characterized by a different integrin profile, differentiation grade and in vitro invasive ability. The levels of total protein kinase C activity revealed a direct correlation with the chemotactic and invasive ability of these clones. Protein kinase C inhibitors, sphingosine and staurosporine, reduced chemotaxis and invasiveness of the highly invasive clone 2/60, while 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H7) was ineffective. Immunofluorescence analysis revealed high levels of protein kinase C α in clone 2/60, while the less invasive clone 2/21 expressed low levels of protein kinase C α and β but surprisingly appreciable levels of protein kinase C γ Downregulation with phorbol 12‐myristate 13‐acetate(TPA) did not affect invasiveness of clone 2/60 unless the compound was present during the assay. H7 strongly increased invasiveness of clone 2/21 and was able to reverse the inhibitory effect of TPA on clone 2/60. Preliminary experiments showed higher levels of diacylglycerol in clones with lower protein kinase C, suggesting a constitutive down regulation of the enzyme in low invasive clones. Our results support a role for protein kinase C in the invasion process, but point out the complexity of the mechanism which might involve the proteolytic fragment of the enzyme, protein kinase M. © 1994 Wiley‐Liss, Inc.
Immunolocalization of granulocyte‐colony‐stimulating factor in human glial and primitive neuroectodermal tumors
Authors: Stan A.‐C. , Walter G. F. , Welte K. , Pietsch T.
Published: February 2007

Granulocyte‐colony‐stimulating factor (G‐CSF) is a hematopoietic cytokine that regulates the differentiation of myeloid progenitors and the function of mature neutrophils. It is produced in vitro by monocytes/macrophages, mesothelial cells, fibroblasts and endothelial cells after appropriate induction by inflammatory mediators like IL‐I and TNF. Normal as well as tumorous glial cells can also be induced to produce CSFs in vitro. However, little is yet known about the in vivo expression of G‐CSF as a mediator in inflammation and malignancy withiin the human central nervous system. The aim of the present study was to investigate by immunostaining the expression of the G‐CSF protein within non‐tumorous and tumorous glial tissues, and primitive neuroectodermal tumors. Using the murine monoclonal anti‐G‐CSF TM 82/60 antibody, we found high G‐CSF expression in astrocytoma WHO grades I and II and reactive brain tissue, low expression in astrocytoma WHO grade III, and none in glioblastoma, oligodendroglioma WHO grades II and III, none medulloblastoma. In consecutive sections of the tissue samples, G‐CSF protein was localized in GFAP‐positive glial cells, but not in macrophages/microglial cells, which expressed HLA‐DR, detected by the antibody CR3/43. Computer‐assisted microdensitometric evaluation of the intensity of immunostaining for G‐CSF and statistic analysis of the data revealed significant differences between the diagnostic entities studied (p < 0.0001). We conclude that in vivo expression of G‐CSF is a characteristic of reactive as well as tumorous astrocytes, with the latter losing this feature at higher degrees of dedifferentiation. © 1994 Wiley‐Liss, Inc.
Cyclin D1 protein expression and function in human breast cancer
Authors: Bartkova Jirina , Lukas Jiri , Müller Heiko , Lützhøt Dorrit , Strauss Michael , Bartek Jiri
Published: February 2007

Cyclin D1 is a cell‐cycle regulator essential for G1, phase progression and a candidate proto‐oncogene implicated in pathogenesis of several human tumour types, including breast carcinomas. In spite of the accumulating genetic evidence, however, there are no data regarding abundance and properties of the cyclin D1 protein in breast cancer. We now report aberrant nuclear overexpression/accumulation of the cyclin D1 protein in about half of the 170 primary breast carcinoma specimens analyzed by monoclonal antibody immunohistochemistry, indicating that the frequency of cyclin D1 abnormalities may be considerably higher than previously deduced from DNA amplification studies. A comparison of the expression patterns in matched lesions at different stages of tumour progression revealed that the cyclin D1 protein aberration appears to reflect a relatively early event and that, when acquired by a tumour, it is maintained throughout breast cancer progression including metastatic spread. In both tumour tissues and breast cancer cell lines, the abundance of this protein shows characteristic variations consistent with a cell‐cycle oscillation and the peak levels expressed in G1. In all 7 cell lines whose retinoblastoma (Rb) protein is mutant or complexed to SV40 T antigen, exceptionally low levels of cyclin D1 protein and mRNA were found. Antibody‐mediated and anti‐sense oligonucleotide knockout experiments demonstrate the requirement for the cell‐cycle regulatory function of cyclin D1 in breast cancer lines with single or multiple copies of the gene and reveal the absence of such a requirement in the cell lines with Rb defects. Our data are consistent with the notion that the emerging “Rb‐cyclin D1 pathway” represents a frequent target of oncogenic abnormalities in breast cancer. © 1994 Wiley‐Liss, Inc.
Study of the resistance of tumor‐cell spheroids to penetration and lysis by activated effector cells
Authors: Ochalek Tomasz , von Kleist Sabine
Published: February 2007

Tumor spheroids and growing cell monolayers were used as 2‐ and 3‐dimensional targets in the in vitro study of the interactions of colorectal tumor cells with various in vitro II‐2‐activated lymphocyte effectors. The study examined tumor‐cell susceptibility to the cytotoxic activity of effector cells in correlation with the ability of the effectors to infiltrate spheroids. No restriction of lymphocyte activity was found when the susceptibility of tumor cells to the panel of autologous and allogeneic II‐2‐stimulated PBL (LAK cells) and TIL was compared. Their activity against a given tumor specimen was similar, but differed against the various other tumor specimens. Thus, the tumor susceptibility and not the cytotoxic potential of the lymphocytes determined the interaction result. An inhibition of lymphocyte penetration was observed in some tumor spheroids, which considerably influenced the resistance to lymphocyte lysis. Apparently it is this inhibition of the migratory capacity of the effector cells by tumor cells that is responsible for tumor resistance to lymphocyte attack. © 1994 Wiley‐Liss, Inc.
Internalization and degradation of monoclonal antibody chCE7 by human neuroblastoma cells
Authors: Novak‐Hofer Ilse , Amstutz Hans Peter , Morgenthaler Jean‐Jacques , Schubiger P. August
Published: February 2007

Internalization and cellular degradation of a chimeric monoclonal anti‐neuroblastoma antibody (MAb chCE7) is described. Immunofluorescence localization showed temperature‐dependent redistribution of MAb chCE7 from the cell surface at O°C to vesicular structures after heating to 37°C. SKN‐AS cells which were pulse‐labelled at O°C with radioiodinated chCE7 released 50% of the initial cell‐bound radioactivity into the medium after 20 hr at 37°C. Low‐molecular‐weight radioactivity in the medium was identified as iodotyrosine and the major intracellular degradation product was found to be an antibody fragment of 43 kDa. Degradation was blocked by chloroquine, an inhibitor of lysosomal function. MAb chCE7 binds to a carbohydrate epitope, as treatment with tunicamycin abolishes cell binding. In adherent cells in culture, inhibition of protein synthesis by cycloheximide affected cell‐surface binding sites for chCE7 in a biphasic manner, with an initial increase followed by long‐term decrease. Expression of binding sites was also found to be inhibited by sodium azide, by the lysosomotropic drug chloroquine and by Brefeldin A, a drug which prevents export of newly synthetized proteins to the cell surface. When cells were treated with high doses of chCE7 up to 24 hr and cell‐surface binding sites were then measured after an acidic buffer wash, no loss of surface binding sites was found, indicating that pretreatment with MAb chCE7 does not induce down‐regulation of its binding sites. © 1994 Wiley‐Liss, Inc.
DCC gene alteration in human endometrial carcinomas
Authors: Gima Tomonao , Kato Hidenori , Honda Tsuyoshi , Imamura Toshiro , Sasazuki Takehiko , Wake Norio
Published: February 2007

The present study was undertaken to define the gene(s) of importance on the long arm of chromosome 18 (chromosome 18q) in endometrial carcinomas. We analyzed loss of heterozygosity (LOH) at 3 loci on chromosome 18q and DCC gene expression by the reverse‐transcriptase/polymerase chain reaction (RT‐PCR) method. Among 61 tumors that were informative, 16 (26%), estimated to be a minimum number, showed allelic losses at one or more chromosome 18q loci. Deletions in these tumors possibly involved the region within or near the chromosome 18q 21.3 band where the DCC gene was localized. Moreover, the incidence of altered DCC mRNA expression was high in these tumors. Appropriate transcription was lost in 5 of 7 (71%) carcinoma cell lines in addition to 14 of 28 (50%) surgically resected tumors. Histopathological differentiation and clinical stage of disease were not related to LOH frequency or to DCC mRNA expression. These results suggest that the target for allelic loss on chromosome 18q seen in endometrial carcinomas is the DCC gene, and that inactivation of this gene may be critical for the development of most endometrial carcinomas. © 1994 Wiley‐Liss, Inc.
Association among p53 gene mutation, nuclear accumulation of the p53 protein and aggressive phenotypes in breast cancer
Authors: Tsuda Hitoshi , Hirohashi Setsuo
Published: February 2007

In order to reveal whether differences in the type and site of p53 gene mutations influence the function of the gene and tumor phenotype, we examined nuclear accumulation of the p53 protein immunohistochemically, loss of the other p53 allele by restriction‐fragment‐length polymorphism analysis, and histological grade of atypia in 52 breast‐cancer tissue specimens in which the position and pattern of the mutation were identified. When mis‐sense point mutations ordeletionsof3nbases(n = 1, 2,…), which did not cause a frame shift downstream, occurred within codons 110‐180 or 234‐285, containing highly conserved regions, the p53 protein was almost always (92%) accumulated in nuclei in a majority of the cancer cells. When these mutations occurred outside these regions, only 46% of the cases showed nuclear accumulation of the protein in a majority of cancer cells. In tumors with non‐sense point mutations or deletion of 3n+1 or 3n + 2 bases (n = 0, 1, 2,…), which caused a downstream frameshift, nuclear accumulation of the p53 protein was absent in 93% of cases. Irrespective of the mutation site or pattern, a majority of cases showing p53 mutation revealed loss of hetero‐zygosity on 17p13 (83%), which suggested they do not carry wild‐type p53 allele, and the highest histological grade of atypia (90%). Regardless of differences in their protein‐expression pattern, a majority of the p53 gene mutations were suggested to function in a recessive mode and to be involved in the development of histologically aggressive breast cancer. © 1994 Wiley‐Liss, Inc.
Oxygen consumption rate and mitochondrial density in human melanoma monolayer cultures and multicellular spheroids
Authors: Hystad Marit E. , Rofstad Einar K.
Published: February 2007

Rate of oxygen consumption per cell has been shown in previous studies to decrease with increasing depth in the viable rim of multicellular spheroids initiated from rodent cells, human colon‐carcinoma cells, and human glioma cells, due to progressive accumulation of quiescent cells during spheroid growth. The purpose of our work was to determine oxygen‐consumption profiles in human melanoma spheroids. Monolayer cultures of 4 lines (BEX‐c, COX‐c, SAX‐c, and WIX‐c) and spheroid cultures of 2 lines (BEX‐c and WIX‐c) were subjected to investigation. Spheroids were initiated from monolayer cell cultures and grown in spinner flasks. Rate of oxygen consumption was measured with a Clarke‐type electrode. Mitochondrial density was determined by stereological analysis of transmission electron micrographs. Thickness of viable rim and cell packing density were assessed by light microscopy of central spheroid sections. Cell‐cycle distribution was determined by analysis of DNA histograms measured by flow cytometry. Cell volume was measured by an electronic particle counter. Rate of oxygen consumption per cell differed by a factor of approximately 1.8 between the 4 cell lines and was positively correlated to total volume of mitochondria per cell. Rate of oxygen consumption per cell and total volume of mitochondria per cell were equal for monolayer cell cultures, 600‐μm spheroids and 1,200‐μm spheroids of the same line. Mitochondrial density and location in the cell did not differ between cells at the spheroid surface, in the middle of the viable rim and adjacent to the central necrosis. Cell‐cycle distribution, cell volume, and cell‐packing density in the outer and inner halves of the viable rim were not significantly different. Consequently, the rate of oxygen consumption per cell in inner regions of the viable rim was probably equal to that at the spheroid surface, suggesting that oxygen diffusion distances may be shorter in some melanomas than in many other tumor types. © 1994 Wiley‐Liss, Inc.
A cell surface antigen (BAL) defined by a mouse monoclonal antibody inducing apoptosis in a human lymphocytic leukemia cell line
Authors: Wallén‐Öhman Marie , Borrebaeck Carl A. K.
Published: February 2007

The lack of apoptosis or programmed cell death in human tumor cells has been suggested to be one factor allowing uncontrolled growth of neoplasms. We have developed a mouse monoclonal antibody (MAb) that induces programmed cell death in a human acute leukemia cell line (KM‐3) of the pre B‐cell type. Stable, antibody‐producing hybridomas were produced by fusing mouse myeloma cells to spleen cells from mice immunized with viable KM‐3 cells. Incubation of KM‐3 cells with the MAb (designated anti‐BAL) resulted in growth inhibition and subsequent cell death within 2‐3 days. Anti‐BAL required cross‐linking with a rabbit anti‐mouse antibody to induce DNA fragmentation typical of apoptosis. Immunoblotting experiments with anti‐BAL identified a 37‐kDa protein, apparently different from any previously described apoptosis‐related surface antigen. Strongest expression of the antigen was generally found on cells of lymphoid or myeloid origin. However, several other cell types such as fibroblasts and endothelial cells were also stained by anti‐BAL in flow cytometry but less intensively. Despite the apparent presence of this cell surface‐bound 37‐kDa antigen on several normal and malignant cell types, anti‐BAL induced cell death only in human malignant cell lines expressing a more immature phenotype. © 1994 Wiley‐Liss, Inc.
Expression of uPA and its receptor by both neoplastic and stromal cells during xenograft invasion
Authors: Rømer John , Pyke Charles , Lund Leif R. , Eriksen Jens , Kristensen Peter , Rønne Ebbe , Høyer‐Hansen Gunilla , Danø Keld , Brünner Nils
Published: February 2007

Recent studies have shown that molecules involved in generation and regulation of extracellular proteolytic activity are often expressed by non‐malignant stromal cells during human cancer invasion. We have studied the expression of the urokinase‐type plasminogen activator and the urokinase‐type plasminogen activator cell‐surface receptor in xenografts of human MDA‐MB‐231 mammary carcinoma cells growing invasively in nude mice. Northern analysis showed the presence of both human and mouse urokinase‐type plasminogen activator and urokinase‐type plasminogen activator receptor mRNA in tumor extracts. By in situ hybridization, mRNA for human urokinase‐type plasminogen activator and its receptor was detected in virtually all the cancer cells, while mouse urokinase‐type plasminogen activator and urokinase‐type plasminogen activator receptor mRNA was expressed by tumor‐infiltrating fibroblast‐like and macrophage‐like cells. In invasive areas the cells expressing the 2 murine mRNAs were either the same or located immediately adjacent to each other. This model system has several advantages for studies of the mechanism by which cancer cells induce or recruit stromal cells to produce molecules involved in proteolysis. © 1994 Wiley‐Liss, Inc.
Doxorubicin retention and chemoresistance in human mesothelioma cell lines
Authors: Isobe Hiroshi , Wellham Larry , Sauerteig Antonieta , Sridhar Kasi S. , Ramachandran Cheppail , Krishan Awtar
Published: February 2007

Eight cell lines were established from the pleural effusion of 4 patients with malignant mesothelioma. The most sensitive (FCCMES‐4) and the most resistant (FCCMES‐2) mesothelioma cell lines had IC50 of 0.66 and 1.85 μM for doxorubicin in clonogenic assays, respectively. In comparison with murine leukemic P388 cells, mesothelioma cell lines were 7.5‐ to 21 ‐fold more resistant to doxorubicin. Co‐incubation with verapamil significantly increased doxorubicin retention in one of the cell lines (FCCMES‐2) expressing P‐glycoprotein in 16.8% of the cells. These results indicate that doxorubicin resistance may be intrinsic in refractory mesothelioma patients and P‐glycoprotein‐mediated drug efflux may be involved in resistance of some of the mesotheliomas. © 1994 Wiley‐Liss, Inc.
Anti‐idiotypic monoclonal antibody AB3, reacting with the primary antigen (CEA), can localize in human colon‐carcinoma xenografts as efficiently as AB1
Authors: De Moraes Jane Z. , Gesztesi Jean‐Luc , Westermann Patrick , Le Doussal Jean‐Marc , Lopes José D. , Mach Jean‐Pierre
Published: February 2007

BALB/c mice were immunized with anti‐idiotypic monoclonal (MAb) antibody (anti‐Id or Ab2) directed against an ABI Mab anti‐carcinoembryonic (CEA) in order to obtain AB3 MAbs (anti‐anti‐Id). AB3 MAbs were shown to recognise the primary antigen (CEA) and one of them was tested extensively in vitro and in vivo. This AB3 MAb was shown to bind specifically to CEA on frozen sections of a human colon carcinoma by immunoper‐oxidase. Scatchard plot analyses showed that the affinity of this AB3 was of the same order of magnitude as the ABI. In vivo experiments, in nude mice bearing CEA‐producing human colon‐carcinoma xenografts showed that up to 30% of the intravenously injected dose of 125I‐labelled AB3 were localized per gram of tumour tissue. Furthermore, calculation of the ratios of AB3 concentration in the tumour over those in normal organs such as lung, liver, kidney, spleen and bone gave relatively high values similar to results obtained with ABI. All together our results show that AB3 can localize as efficiently and specifically in the tumour as ABI, despite the fact that the mice from which it was derived were immunized with a mouse MAb (AB2) and had never been exposed to CEA. © 1994 Wiley‐Liss, Inc.
The induction of apoptosis is a common feature of the cytotoxic action of ether‐linked glycerophospholipids in human leukemic cells
Authors: Diombde Luisa , Piovani Bianca , Re Fabio , Principe Paola , Colotta Francesco , Modest Edward J. , Salmona Mario
Published: February 2007

The ability of 2 recent ether‐lipid derivatives, aza‐phospholipids BN52205 and BN522II, to induce apoptosis in different leukemia cell lines was investigated using I‐octadecyl‐2‐methly‐rac‐glycero‐3‐phosphocholine (ET‐18‐OCH3) as a positive control. HL60, K562, Molt‐4 and U937 cells were exposed for 24 hr to 20 μM of drug. The 2 aza‐derivatives were as cytotoxic as ET‐18‐OCH3: BN52205 and BN522II selectively induced apoptotic death in HL60, Molt‐4 and U937 cells, but not in the KS62‐resistant cell line. Around 50% of DNA was fragmented in HL60 cells after exposure to the aza‐derivatives, and 34% and 20% of DNA was fragmented in Molt‐4 and U937 cells respectively. Similar results were obtained when cells were exposed to ET‐18‐OCH3. Our data confirm that ether lipids induce apoptosis in a variety of human leukemic cells, providing a possible explanation for their selectivity and mechanism of action.
Establishment and in vivo characterization of multidrug‐resistant dunning R3327 rat prostate‐carcinoma cell‐lines
Authors: Bashir Imtiaz , Sikora Karol , Abel Paul , Foster Christopher S.
Published: February 2007

We describe the selection of 3 new multidrug‐resistant cell lines derived from tumor cells of different metastatic phenotypes within the Dunning R3327 model of rat prostatic carcinoma. Cell lines of weak (AT2) and strong (AT3 and MAT‐LyLu) metastatic behavior were cultured in vitro and challenged with doxorubicin at progressively increasing concentrations. Chemosensitivity was determined colorimetrically by release of precipitated formazan pigment (MTT assay). Expression of the multidrug‐resistance glycoprotein (P‐170) was monitored immunocytochemically and by Western blotting using monoclonal antibody C219. The behavior of the parental and resultant drug‐resistant cells was assessed by their growth in syngeneic rats. Doxorubicin challenge of the initially drug‐sensitive parental prostatic carcinoma cell lines resulted in the rapid development of multidrug resistance together with simultaneous expression of P‐glycoprotein. While lung and lymph‐node metastases developed in host animals inoculated with parental AT3 and MAT‐LyLu cells, no metastases developed in the multidrug‐resistant progeny of these cell lines. This study has shown that Dunning rat prostate‐carcinoma cell lines, previously sensitive to different cytotoxic agents, rapidly become multidrug‐resistant and express P‐glycoprotein following exposure to doxorubicin. Further more, development of multidrug resistance is associated with a less aggressive tumor phenotype and loss of metastatic potential. Nevertheless, it is unlikely that the non‐metastatic phenotype of Dunning rat prostatic carcinoma cells is solely associated with expression of P‐glycoprotein. These new multidrug‐resistant cell lines exhibiting an altered behavioral phenotype will provide a valuable mode with which to analyze the relationship between expression of P‐glycoprotein and the metastatic phenotype of prostatic carcinoma cells.
Inhibition of metastasis of lewis lung carcinoma by a synthetic peptide within growth factor‐like domain of urokinase in the experimental and spontaneous metastasis model
Authors: Kobayashi Hiroshi , Gotoh Junko , Fujie Michio , Shinohara Hiromitsu , Moniwa Nobuhiko , Terao Toshihiko
Published: February 2007

Four synthetic peptides (residues 20‐30 and 17‐34) within the growth factor‐like domain (GFD) of murine and human urokinase‐type plasminogen activator (uPA) were examined to determine whether they inhibit production of experimental and spontaneous lung metastasis by murine Lewis lung carcinoma (3LL) cells. In an in vivo experimental metastasis assay, which determines mainly the later steps of the metastatic migration process (extravasation from the bloodstream and then growth into pulmonary tumor), none of the peptides introduced by i.v. single co‐injection into syngeneic C57B1/6 mice inhibited pulmonary metastasis, when 3LL cells were pre‐incubated with the peptides followed by i.v. co‐injection of the peptide and cells. In addition, none of the peptides, when injected i.p. daily for 7 days after i.v. tumor cell inoculation, reduced the number of lung tumor colonies. In a second in vivo assay that measures metastasis from a primary tumor (spontaneous metastasis model), multiple i.p. injections of the mouse peptide 17‐34 for 7 days after s.c. tumor cell inoculation significantly inhibited metastatic lung tumor colonization in a dose‐dependent manner, whereas human peptide 17‐34 had no effect. Mouse and human peptide 20‐30 had no effect either. The inhibition of lung metastasis was not due to direct antitumor effects of mouse peptide 17‐34. Our results indicate that occupation of uPA receptors on 3LL cells by the enzymatically inactive mouse peptide 17‐34 or prevention of rebinding of uPA synthesized by tumor cells to their receptor specifically reduced tumor cell invasion and formation of metastasis and that uPA may regulate more efficiently the mechanism involved in the entry of tumor cells into vascular circulation than extravasation during the metastatic process.
T‐helper‐ and accessory‐cell‐independent cytotoxic responses to human tumor cells transfected with A B7 retroviral vector
Authors: Döhring Christian , Angman Lena , Spagnoli Giulio , Lanzavechia Antonio
Published: February 2007

As a means to increase the immunogenicity of tumor cells, we have developed a retroviral vector to transfect human B7, a molecule capable of delivering co‐stimulatory signals to T cells. Three different tumors, a melanoma, an ovarian carcinoma and a myelomonocytic leukemia, were transfected with high efficiency. When compared for their capacity to stimulate allogeneic T cells, B7+ but not B7− tumor cells were able to stimulate strong proliferative and cytotoxic responses. The effector CTL generated recognised B7+ and B7− cells as well as untransfected tumor cells, indicating that B7 is required in the inductive but not the effector phase of the response. Remarkably, B7+ tumor cells were able to induce cytotoxic responses both by CD4‐depleted and by CD8‐purified T cells, demonstrating that expression of B7 is at the same time necessary and sufficient to induce a cytotoxic response in the absence of T‐helper cells and accessory cells.
Esophageal balloon cytology and subsequent risk of esophageal and gastric‐cardia cancer in a high‐risk chinese population
Authors: Liu S. F. , Shen Q. , Dawsey S. M. , Wang G. Q. , Nieberg R. K. , Wang Z. Y. , Weiner M. , Zhou B. , Cao J. , Yu Y. , Guo W. D. , Li J. Y. , Blot W. J. , Li B. , Taylor P. R.
Published: February 2007

Linxian, China has some of the highest rates of esophageal/gastric cardia cancer in the world. In 1983, esophageal balloon cytology screening was performed in 3 communes in northern Linxian. Of the participants, 10,066 with no evidence of cancer were followed prospectively for 71/2 years to evaluate the ability of the initial cytologc diagnoses to identify individuals at increased risk for developing cancer of the esophagus or gastric cardia. A total of 747 incident cases of esophageal or cardia cancer and 322 deaths due to these tumors were identified during the follow‐up period and used in this analysis. The risks for esophageal or cardia cancer incidence and mortality increased in parallel with the presumed severity of the 1983 Chinese cytologic diagnoses. After adjusting for potential confounding factors, relative risks for esophageal or cardia cancer incidence, by initial cytologic diagnosis, were normal = 1.00 (reference), hyperplasia = 1.25, dysplasia 1 = 2.20, dysplasia 2 = 4.22 and near‐cancer = 5.96. Our results suggest that esophageal balloon cytology, as performed and interpreted in Linxian in 1983, successfully identified individuals at increased risk for developing cancer of the esophagus or gastric cardia. © 1994 Wiley‐Liss, Inc.
Enhancement of radioimmunotherapy by drugs modifying tumour blood flow in a colonic xenograft model
Authors: Pedley R. Barbara , Begent Richard H. J. , Boden Joan A. , Boxer Geoffrey M. , Boden Robert , Keep Patricia A.
Published: February 2007

Radioimmunotherapy (RIT) is hampered clinically by poor tumour localization of antibody. In order to enhance localization we have investigated the concomitant use of RIT with 2 drugs, fiavone‐8‐acetic acid (FAA) and its analogue 5,6‐dimethylxanthenone‐4‐acetic acid (XAA), which both reduce tumour blood flow and induce immunomodulation. A single i. v. dose of 0.5 mCi (18.5 MBq) intact 131 I anti‐CEA antibody significantly delayed growth and prolonged survival over that of untreated controls, in an established LSI74T colon xenograft model in nude mice. The adjuvant use of a single i. p. dose of either FAA or XAA, given 24 or 48 hr after 131 I‐A5B7 to allow maximum tumour levels of antibody to be attained before drug‐induced blood‐flow inhibition, significantly enhanced the RIT. FAA caused entrapment of antibody within the tumour in relation to the time allowed for localization before drug administration. Repeated doses of FAA prolonged tumour growth inhibition but did not enhance the therapy achieved after a single dose. Although both drugs alone induced massive tumour necrosis of all but a thin peripheral rim of viable cells, tumour regrowth was inhibited for a few days only, with no effect on survival. Drug‐induced tumour necrosis, immunomodulation and retention of higher doses of 131 I‐A5B7 within the tumour may contribute to the enhanced RIT produced by this combined therapy. © 1994 Wiley‐Liss, Inc.
Anti‐Tac(Fab)‐PE40, a recombinant double‐chain immunotoxin which kills interleukin‐2‐receptor‐bearing cells and induces complete remission in an in vivo tumor model
Authors: Kreitman Robert J. , Chang Chung Nan , Hudson Dehra V. , Queen Cary , Bailon Pascal , Pastan Ira
Published: February 2007

We have produced a single plasmid encoding both the heavy chain Fd domain (VH + CHl) of the anti‐interleukin‐2 receptor (IL2R) monoclonal antibody anti‐Tac, and the anti‐Tac light chain fused to PE40, a truncated derivative of Pseudomonas exotoxin. The active immunotoxin anti‐Tac(Fab)‐PE40 could be recovered from E. coli from either periplasm or renatured inclusion bodies. The double‐chain immunotoxin was very cytotoxic toward IL2R‐bearing cell lines, human activated T cells and fresh adult‐T‐cell‐leukemia cells. The cytotoxicity was similar to that of anti‐Tac(Fv)‐PE40, the single‐chain recombinant toxin containing only the variable domains of anti‐Tac. IL2R‐binding affinity was also equivalent to that of anti‐Tac(Fv)‐PE40, which is one‐third that of anti‐Tac. The serum half‐life in mice was significantly prolonged as compared with anti‐Tac(Fv)‐PE40, with a β phase of 430 vs. 57 minutes, but the LD50s were equivalent when the immunotoxins were administered in 3 daily doses. Anti‐Tac(Fab)‐PE40 was very cytotoxic in vitro toward transfected ATAC‐4 carcinoma cells which express IL2Rs. In mice bearing ATAC‐4 tumors, anti‐Tac(Fab)‐PE40 showed significant anti‐tumor activity, inducing complete remissions in 80 and 100% of treated animals at approximately 7 and 14% respectively of the LD50. Anti‐Tac(Fab)‐PE40 was much more effective in vitro and in vivo than chemical conjugates between anti‐Tac and truncated PE molecules. The recombinant Fab toxin should be studied further as potential treatment for IL2R‐related malignancies, particularly if smaller recombinant immunotoxins have insufficient half‐life in humans. © 1994 Wiley‐Liss, Inc.
Analysis of proviral integration in human mammary epithelial cell lines immortalized by retroviral infection with a temperature‐sensitive SV40 T‐antigen construct
Authors: Stamps Alasdair C. , Davies Susan C. , Burman Jacqueline , O'Hare Michael J.
Published: February 2007

A panel of eight conditionally immortal lines derived by infection of human breast epithelial cells with an amphotropic retrovirus transducing a ts mutant of SV40 large T‐antigen was analyzed with respect to individual retroviral integration patterns. Each line contained multiple integration sites which were clonal and stable over extended passage. Similar integration patterns were observed between individual lines arising separately from the same stock of pre‐immortal cells, suggesting a common progenitor. Retroviral integration analysis of preimmortal cells at different stages of pre‐crisis growth showed changes indicative of a progressive transition from polyclonality to clonality as the cells approached crisis. Each of the immortal lines contained a sub‐set of the integration sites of their pre‐immortal progenitors, with individual combinations and copy numbers of sites. Since all the cell lines appeared to originate from single foci in separate flasks, it is likely that each set arose from a common clone of pre‐immortal cells as the result of separate genetic events. There was no evidence from this analysis to suggest that specific integration sites played any part either in the selection of pre‐crisis clones or in the subsequent establishment of immortal lines. © 1994 Wiley‐Liss, Inc.
The state of differentiation of HT‐29 colon carcinoma cells alters the secretion of cathepsin D and of plasminogen activator
Authors: Huet Guillemette , Zerimech Farid , Dieu Marie‐Claire , Hemon Brigitte , Grard Georges , Balduyck Malika , Janin Anne , Lafyatis Robert , Degand Pierre
Published: February 2007

We have studied the cellular content and the extracellular release of cathepsins B and D, and of plasminogen activator, in 2 different tumor cell populations before confluence and after late confluence: the HT‐29 colon carcinoma cell line, which contains primarily undifferentiated cells, and a subpopulation derived from this cell line, which contains cells committed to differentiation into mucus‐secreting goblet cells after confluence. In both populations, cellular cathepsin‐B activity increased after confluence, and latent cathepsin B was found in all culture media. In the parental cell line, cellular cathepsin D activity decreased after confluence; however, cathepsin D was secreted at high levels into the extracellular medium. In contrast, in the subpopulation of cells committed to differentiation, cellular cathepsin D activity increased after confluence, and cathepsin D was not secreted into the extracellular medium, but was immunolocalized in the apical brush border of the differentiated cells. Plasminogen activator of urokinase type was identified by immunocytochemistry. Both subconfluent cell populations, and the post‐confluent undifferentiated cell population, produced plasminogen activator activity at similar levels. In contrast, in the differentiated postconfluent cells, the production of plasminogen activator activity was markedly lower. Our data show that the differentiation of HT‐29 colon carcinoma cells into mucus‐secreting cells impairs the secretion of plasminogen activator and cathepsin D, but does not affect cathepsin B. © 1994 Wiley‐Liss, Inc.
Evaluation of a new photosensitizer, meso‐tetra‐hydroxyphenyl‐chlorin, for use in photodynamic therapy: A comparison of its photobiological properties with those of two other photosensitizers
Authors: Ma Liwei , Moan Johan , Berg Kristian
Published: February 2007

The properties of a new photosensitizer, meso‐tetra‐hydroxyphenyl‐chlorin (mTHPC), were studied using V79 cells (Chinese‐hamster lung fibroblasts). Comparisons were made with 2 other photosensitizers: photofrin II (PII) and meso‐tetra‐hydroxyphenyl‐porphyrin (mTHPP). A main advantage of mTHPC is that it has a strong absorption at 652 nm. Maximal cellular uptake of the dye was observed after 24 hr incubation of the cells with the drug. Using a confocal laser‐scanning fluorescence microscope, we observed a diffuse distribution of m THPC in the cytoplasm. Furthermore, the lipophilicity of mTHPC was compared with that of the components of PII by means of high‐pressure liquid chromatography (HPLC). Absorption and fluorescence spectroscopy indicated that aggregated as well as monomeric mTHPC was bound to the cells. The action spectrum for photo‐inactivation of the cells showed that aggregated mTHPC did not contribute significantly to its photosensitizing effects. In the present cellular system, the efficiency of photodynamic therapy (PDT) with mTHPC (cells were irradiated at a wavelength of 652 nm) was higher than with PII (irradiation at 630 nm) or with mTHPP (648 nm). The quantum yield for photo‐inactivation of cells was smaller for mTHPC than for mTHPP and PII. The addition of 1,3‐diphenylisobenzofuran (DPBF) reduced cell inactivation during PDT. Thus, PDT with mTHPC seems to act at least partlyviaa type‐II process. © 1994 Wiley‐Liss, Inc.
Caffeine attenuates the action of amsacrine and etoposide in U‐937 cells by mechanisms which involve inhibition of RNA synthesis
Authors: Pérez Concepción , Pelayo Francisco , Vilaboa Nuria E. , Aller Patricio
Published: February 2007

Pulse treatments of U‐937 human promonocytic leukemia cells with the DNA topoisomerase‐II inhibitors 4′‐(9‐acridynilamino)methanesulfon‐m‐anisidide (amsacrine, mAMSA) or etoposide (VP‐16) caused growth inhibition, G2‐arrest, increase in cell size and expression of differentiation markers. All these effects were greatly reduced by the presence of 5–10 mM caffeine. In addition, caffeine partially prevented the increase in the number of topoisomerase‐DNA cleavable complexes caused by the topoisomerase inhibitors, as determined by SDS/CIK precipitation assays; it caused chromatin condensation, as determined by flow cytometry assays, and interacted with mAMSA in solution, as suggested by spectrophotometric assays. Pulse treatment with caffeine greatly inhibited RNA synthesis but not DNA or protein synthesis, as indicated by labelled precursor incorporation assays. The transcription inhibitor 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzymidazole reduced the mAMSA‐ and VP‐16‐produced growth inhibition in a similar manner. It is concluded that RNA synthesis inhibition is one of the possible mechanisms by which caffeine protects cells from the action of topoisomerase‐II inhibitors. © 1994 Wiley‐Liss, Inc.
Aberrant RB gene expression in routinely processed, archival tumor tissues determined by three different anti‐RB antibodies
Authors: Geradts Joseph , Hu Shi‐Xue , Lincoln Clinton E. , Benedict William F. , Xu Hong‐Ji
Published: February 2007

The retinoblastoma (RB) susceptibility gene encodes a nuclear phosphoprotein which is likely involved in cell cycle control and cell differentiation. The RB protein is mutated or absent in a variety of human malignancies. Its role as a molecular marker for clinical tumor behavior is under extensive investigation. However, studies on the status of the RB protein in primary or metastatic tumors and their precursor lesions have been slowed by the lack of availability of a sensitive, reliable assay which allows examination of RB expression in selected cell populations within archival tissues. Thus far, meaningful immunohistochemical analysis of RB protein in formalin‐fixed, paraffin‐embedded specimens has been achieved only with the polyclonal antibody RB‐WL‐1. We now describe a method which produces excellent staining results in formalin‐fixed, routinely processed tissues, using commercially available monoclonal antibodies (MAbs) in conjunction with an antigen‐retrieval step. The resulting stains were superior to those on frozen sections and comparable to those obtained with RB‐WL‐1. Twelve of 51 random invasive bladder cancers (24%) had abnormal expression of the RB gene, as determined by immunohistochemistry. Smaller cohorts of breast, prostate and lung carcinomas had incidences of aberrant RB gene expression ranging from 9% to 24%. Since the staining method was widely applicable to essentially all formalin‐fixed, archival tissues, it may expedite studies on the biological and clinical significance of altered RB expression in human neoplasia.
Risk factors for esophageal cancer in Shanghai, China. I. Role of cigarette smoking and alcohol drinking
Authors: Gao Yu‐Tang , McLaughlin Joseph K. , Blot William J. , Ji Bu‐Tian , Benichou Jacques , Dai Qi , Fraumeni Joseph F.
Published: February 2007

A population‐based case‐control study of esophageal cancer was conducted in urban Shanghai involving interviews with 902 cases and 1,552 controls. Risk of esophageal cancer was increased among tobacco smokers and alcohol drinkers. Odds ratios (OR) for smoking were 2.1 and 1.6 for men and women, respectively, and increased with number of cigarettes smoked per day, duration of smoking, number of pack‐years and decreasing age at start of smoking. For men who were current alcohol drinkers OR was 1.4, with the excess risk primarily among heavy drinkers. Few women drank alcoholic beverages. The combined effect of heavy smoking and drinking among men was pronounced: OR was 12.0 for those who smoked more than 1 pack per day and drank more than 750 g of ethanol per week. The relation with smoking appeared stronger for cancers of the middle and lower thirds of the esophagus than for the upper third, while patterns of risk for squamous cell carcinoma and adenocarcinoma were similar. Heavy drinking affected all 3 subsites, with increased risks mainly limited to squamous cell carcinoma. Cigarette smoking and alcohol drinking combined accounted for almost 50% of all esophageal cancers among men in Shanghai; among women, 14% of cases were attributed to smoking. Our study confirms that smoking and drinking are important risk factors for esophageal cancer in China, thereby paralleling findings from developed countries.