bluuebunny/crossref_metadata_2025 huggingface dataset:
Target cells are sensitized for cytotoxic T‐lymphocyte‐mediated destruction by estradiol and tamoxifen
Authors: Baral Edward , And Eva Nagy , Berczi Istvan
Published: February 2007

The effect of estradiol (E2) and tamoxifen (TX) on cytotoxic T‐tymphocyte (CTL)‐mediated target‐cell lysis was studied. CTL was generated in mixed cultures of rat spleen cells, using female Fischer 344 rat cells as responders and female Wistar rat cells or Nb2 rat lymphoma cells as stimulators. Concanavalin‐A‐stimulated Wistar lymphoblasts or Nb2 cells were used as targets. CTL harvested on day 5 exerted 16‐25% cytotoxicity when used at 1:12‐1:50 target:effector cell ratios. Day‐6 CTL had no cytotoxic activity. Treatment of target cells with either TX or E2, or both, at I‐μM concentrations for 4 hr prior to cytotoxicity testing raised the target‐cell killing to 100%. Highly significant enhancement of cytotoxicity was also observed when the drugs were used at I00‐, 10‐, or 1‐nM concentrations. Treatment of effector cells under similar conditions led to inhibition of cytotoxicity at I‐μM concentration, some enhancement at 100 nM and no effect at 10 and I nM. When treated target and effector cells were combined, the amplification of target‐cell lysis was similar in magnitude to that seen in tests with treated targets only. Drug treatment of target cells had no influence on their resistance to lysis in hypotonic solutions or on total and spontaneous 51Cr release. The inhibition of DNA and protein synthesis in target cells interfered with both basal cytolysis and drug‐induced enhancement. The release of the nuclear label, [3H]‐thymidine, from Nb2 targets by CTL, was also enhanced by target treatment with TX. These results illustrate that CTL‐mediated cytotoxicity is amplified by physiological concentrations (1 and 10 nM) of E2 and equimolar concentrations of TX. © 1994 Wiley‐Liss, Inc.
Melanoma‐specific CD4+ T lymphocytes recognize human melanoma antigens processed and presented by epstein‐barr virus‐transformed B cells
Authors: Topalian Suzanne L. , Rivoltini Licia , Mancini Marie , Ng Jennifer , Hartzman Robert J. , Rosenberg Steven A.
Published: February 2007

While much emphasis has been placed on the role of MHC class I‐restricted CDS+ T cells in the recognition of tumor‐specific antigens (Ag), evidence has accumulated that CD4+ T cells also play a critical role in the anti‐tumor immune response. However, little information exists on the nature of MHC class II‐restricted human tumor Ag. In an attempt to develop in vitro systems to characterize such Ag, we examined the ability of Epstein‐Barr virus(EBV) transformed B cells to present melanoma‐associated Ag to melanoma‐specific CD4+ cells. CD4+ T cells cultured from lymphocytes infiltrating a s.c. melanoma metastasis secreted TNF‐α and GM‐CSF specifically in response to autologous cultured melanoma cells expressing MHC class II molecules. These CD4+ cells also recognized MHC class II‐compatible EBV‐B cells pulsed with extracts of autologous melanoma cells, but failed to recognize EBV‐B cells pulsed with autologous non‐transformed cells or a variety of allogeneic tumors or normal cells. B cells pre‐fixed with paraformaldehyde were incapable of Ag presentation, suggesting that intracellular processing events were occurring. Antibody‐blocking studies defined HLA‐DR as the dominant if not exclusive restriction locus in this T‐B interaction, and HLA‐DR genocyping revealed DRBI 0404 to be the probable restriction element. In a second patient, a CD4+ T‐cell clone cultured from a melanoma lesion recognized autologous tumor Ag presented by autologous EBV‐B; no cross‐reactivity was observed with the other tumor system investigated, nor with autologous CD4+ T cells specific for tetanus toxoid. These findings demonstrate that tumor Ag can be processed and presented by EBV‐transformed B cells to MHC class II‐restricted tumor‐specific CD4+ T cells. They also provide a model system for direct identification of these tumor‐derived antigens. © 1994 Wiley‐Liss, Inc.
Hydroxyrubicin, a deaminated derivative of doxorubicin, inhibits mammalian DNA topoisomerase II and partially circumvents multidrug resistance
Authors: Solary Eric , Ling Vi‐He , Perez‐Soler Roman , Priebe Waldemar , Pommier Yves
Published: February 2007

In vivo effectiveness of doxorubicin remains restricted due to toxicity and drug resistance. Hydroxyrubicin is a synthetic analog of doxorubicin in which the basic amino group at the C‐3′ has been replaced by a hydroxyl group in order to overcome recognition by the multidrug resistant (MDR) P‐ghlcoprotein and limit cardiotoxicity. The present study shows that hydroxyribicin is a less potent intercalator than doxorubicin. Induction of topoisomerase II‐mediated DNA cleavage in the human C‐myc origin by the two drugs was similar, reaching a maximum at 0.5 μM. Results from the NCI Cell Screening program indicate a relatively good correlation between the cytotoxicity of the 2 drugs on 55 cell lines of various origins (r = 0.723). Using a donogenic assay, we observed that hydroxyrubicin was 20‐fold more cytotoxic against the MDR KB‐VI cell line than doxorubicin and was slightly more cytotoxic than doxorubicin in the sensitive KB3.1 cell line. Uptake studies showed that doxorubicin was retained up to I hr in KB3.1 cells and rapidly eliminated from resistant KB‐VI cells. In contrast, hydroxyrubicin was rapidly eliminated from both sensitive KB3.1 and MDR‐positive KB‐VI cells. Both drugs induced protein‐linked DNA single‐strand breaks (SSBs) in both KB3.I and KB‐VI cells, which is consistent with topoisomerase inhibition. However, the kinetics of DNA SSBs induced by both drugs was very different. DNA breaks disappeared quickly in both KB3.1 and KB‐VI cell lines after hydroxyrubicin removal while DNA breaks induced by doxorubicin disappeared very slowly in KB3.1 cells and rapidly in KB‐VI cells. We conclude that removal of the basic amino group at the C‐3′ of doxorubicin modifies drug transport and partially circumvents MDR without changing topoisomerase II inhibition when compared with doxorubicin. © 1994 Wiley‐Liss, Inc.
Non‐proteolytic release of carcinoembryonic antigen from normal human colonic epithelial cells cultured in collagen gel
Authors: Kinugasa Tetsushi , Kuroki Motomu , Yamanaka Takahiko , Matsuo Yoshino , Oikawa Shinzo , Nakazato Hiroshi , Matsuota Yuji
Published: February 2007

Recent studies have shown that, even with a minimal content of carcinoembryonic antigen (CEA), normal human colonic epithelial cells express substantial amounts of CEA mRNA and colonic mucosal fragments cultured in vitro produce CEA quite actively, indicating that CEA should no longer be considered to be of an oncofetal nature. To understand the basis of the usefulness of CEA as a tumor marker, we analyzed the release of CEA, a glycosyl‐phosphatidylinositol (GPI)‐anchored protein, from colonic epithelial cells, by culturing isolated colonic crypts in collagen gel. The crypts appeared to preserve their morphological and biochemical integrity in the gel for at least 16 hr, and released CEA spontaneously. Three forms of CEA—spontaneously released CEA, CEA liberated with phosphatidylinositol‐specific phospholipase C (PI‐PLC) and CEA in cell lysates—were indistinguishable on SDS‐PAGE. This is in contrast to recombinant CEA spontaneously released from CHO transfectants, which showed a smaller molecular mass than that of PI‐PLC‐cleaved recombinant CEA. By phase separation using Triton X‐114, CEA in the cell lysates of crypts was separated mostly into the detergent phase, while the spontaneously released and the PI‐PLC‐cleaved CEA were separated into the aqueous phase. When the cells were metabolically labeled with the precursors of the GPI‐anchor, 3H‐ethanolamine but not JH‐palmitic acid was found in the spontaneously released CEA. These findings suggest that, in contrast to the proteolysis‐like release of the recombinant CEA from CHO cells, CEA in normal colonic epithelial cells is released by a non‐proteolytic cleavage, which probably occurs through the action of some endogenous phospholipase. © 1994 Wiley‐Liss, Inc.
Transfection of glioma cells with the neural‐cell adhesion molecule NCAM: Effect on glioma‐cell invasion and growth in vivo
Authors: Edvardsen Klaus , Pedersen Paal‐Henning , Bjerkvig Rolf , Hermann Gregers G. , Zeuthen Jesper , Laerum Ole Didrik , Waish Frank S. , Bock Elisabeth
Published: February 2007

The tumor growth and the invasive capacity of a rat glioma cell line (BT4Cn) were studied after transfection with the human transmembrane 140‐kDa isoform of the neural‐cell adhesion molecule, NCAM. After s.c. injection, the NCAM‐transfected cells showed a slower growth rate than the parent cell line (BT4Cn). Upon intracerebral implantation with BT4Cn cells and different clones of NCAM‐transfected cells, all animals developed neurological symptoms within 13‐16 days. However, the tumors showed different growth characteristics. The NCAM‐transfected BT4Cn cells were localized in the region of the injection site, with a sharply demarcated border between the tumor and brain tissue. In contrast, the parental cell line showed single‐cell infiltration and more pronounced destruction of normal brain tissue. Using a 51Cr‐release assay, spleen cells from rats transplanted with BT4Cn tumor cells generally showed a lower cytotoxic response than the spleen cells from rats transplanted with the transfected variants of BT4Cn cells, indicating that the transfection procedure in itself mediated an activation of the immune system. The present data suggest that NCAM may influence the malignant behavior of rat glioma cells in vivo. © 1994 Wiley‐Liss, Inc.
Molecular requirements for rapid plasmacytoma and Pre‐B lymphoma induction by abelson murine leukemia virus in myc‐transgenic mice
Authors: Sugiyama Hiroyuki , Wang Yisong , Jackson Peter , Sawyers Charles L. , Klein George
Published: February 2007

Mutants and fusion products of the c‐abl gene were used to define some of the molecular requirements for rapid plasmacytoma (PC) and pre‐B‐lymphoma induction in pri5tane‐treated N‐myc transgenic BALB/c mice. A‐MuLV induced PCs in 21 of 25 mice with a mean post‐pristane latency period of 46 ± 9 days, compared to 134 ± 25 days in controls exposed to pristane alone. ΔXB, a mutant of type IV c‐abi with a deletion of the SH3 domain, was equally effective in inducing PCs in 7 of 7 mice with a latency period of 49 ± 7 days, indicating that gag sequences are not required for rapid PC induction. The ΔXBΔNar mutant that carried a large C‐terminal deletion in addition showed only a negligible activity, if any, suggesting that PC acceleration requires the C‐terminal domain in the same way as lymphoid transformation and in contrast to fibroblast transformation. BCR‐ABL fusion constructs encoding an 185‐kDa protein as in acute leukemia, or a 210‐kDa protein as in chronic myelocytic leukemia (CML), did not accelerate pristane‐induced PC development in the N‐mye transgenic mice, in contrast to their known ability to immortalize lymphoid cells in vitro. Only one of 14 non‐transgenic littermates developed a pre‐B lymphoma after A‐MuLV infection, and none of 10 normal littermates infected with ΔXB virus developed a construct‐carrying tumor. This result suggests that PC acceleration is due to co‐operative interaction of the N‐myc transgene and activated abl. Infection of N‐myc transgenic bone marrow or spleen cells with A‐MuLV in vitro led to the outgrowth of pre‐B lymphonias after transplantation to pristine‐treated BALB/c recipients. The rymphoma‐inducing activity of A‐MuLV depends on its high titer, since diluted A‐MuLV or the lower‐titered ΔXB induced only PCs under the same conditions. The v‐abl, ΔXB and BCR‐ABL‐carrying viruses generated immortalized lymphoblastoid lines in vitro, regardless of the presence of the N‐myc transgene, suggesting that lymphoid transformation is a direct function of appropriate abl sequences in contrast to PC acceleration.
Characterization of autologous tumor‐specific T‐helper 2 cells in tumor‐infiltrating lymphocytes from a patient with metastatic melanoma
Authors: Kharkevitch Dmitri D. , Seito Dai , Balch Glen C. , Maeda Tomoyuki , Balch Charles M. , Itoh Kyogo
Published: February 2007

Human autologous tumor‐specific T‐helper 2 (Th2) cells were investigated in melanoma tumor‐infiltrating lymphocytes (TILs). Both a CD4+ T‐cell line and its 5 potential T‐cell clones established from TILs of a patient with metastatic melanoma produced significant levels of IL‐4, IL‐6, IL‐10 and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) in response to autologous, but not any of 12 allogeneic, melanoma cell lines. They also produced IL‐3 and IL‐8 but not IL‐2, IFN‐γ, TNF‐α or TNF‐β in response to autologous tumor cells. Furthermore, they showed autologous melanoma‐specific cytotoxicity only in an 18‐hr 51Cr‐release assay. Specific IL‐4, IL‐6 or IL‐10 production by the CD4+ M73 T‐cell line and its clone was inhibited by anti‐class 11 DR (but not anti‐class 1) MAb, whereas their specific cytotoxicity was inhibited by anti‐class 1 (but not anti‐class 11) MAb. Anti‐CD3 and ‐CD4 MAb (but not anti‐CD8) abrogated both IL‐4, IL6 and IL‐10 production and cytotoxicity, while anti‐IL‐4 antibody did not inhibit cytotoxicity. CD4+ potential T‐cell clones, but not CD8+ clones, that were established from freshly isolated TILs without in vitro sensitization by autologous tumor cells also produced IL‐4, IL‐6 and IL‐10 but not IFN‐γ or tumor necrosis factor (TNF)α in an autologous tumor‐specific fashion. These Th2 cells were neither reactive to EBV‐B cells nor suppressive against CD8+ T‐cell clones. PMA and PHA stimulated these potential T‐cell clones, regardless of their specific lymphokine production, to produce IL‐3, IL‐4, IL‐6, IL‐8, IL‐10, GM‐CSF, TNFα and IFN‐γ. Our results demonstrate the presence of autologous tumor‐specific Th2 cells at the melanoma sites.
DNA ploidy in primary fallopian‐tube carcinoma using image cytometry
Authors: Rosen Alexander C. , Graf Anton H. , Klein Matthias , Lahousen Michael , Vavra Norbert , Reiner Angelika , Hacker Gerhard W.
Published: February 2007

Determination of DNA ploidy in 61 samples of primary Fallopian‐tube carcinoma (FTC) by use of image cytometry is presented. Of these samples, 48 showed an aneuploid DNA content. Patients with euploid DNA content showed a median survival time of 33.8 months compared to 24.5 months for aneuploid cases. A high incidence of aneuploid tumors was consistently observed among all FIGO stages and in all groups of histologic grading. However, no correlation between ploidy and FIGO stage or histologic garding could be demonstrated. This observation could be regarded as a marker for the high aggressiveness of FTC which has been repeatedly described in the past.
T lymphocytes infiltrating renal cell carcinoma have a reduced expression of transferrin receptor
Authors: Kudoh Seiji , Stanley Jill , Edinger Mark G. , Tubbs Raymond R. , Klein Eric , Bukowski Ronald M. , Finke James H.
Published: February 2007

T‐cell responses have been reported to be impaired in cancer patients, and lymphocytes infiltrating human tumors (T‐TIL) appear to be more affected than those in the peripheral blood. T‐TIL display a poor proliferative response when compared to peripheral blood T (T‐PBL) cells that show a strong response to all stimuli. Here we report that T‐TIL from patients with renal cell carcinoma (RCC) also have a defect in transferrin receptor (TfR) expression that is not present in T‐PBL cells. Immunocy‐tometry studies (dual staining for CD3s and TfR) demonstrated that autologous T cells from the peripheral blood but not from the tumor expressed TfR following stimulation with IL2, anti‐CD3 or PHA. Expression of TfR correlated with the capacity of T cells from the blood and tumor to proliferate. Gene expression studies using reverse transcriptase‐polymerase chain reaction (RT‐PCR) demonstrated that TfR mRNA levels in T‐TIL were undetectable or low relative to T‐PBL following stimulation. The failure to detect TfR mRNA in T‐TIL after stimulation was not due to a shift in kinetics of mRNA accumulation since TfR mRNA was not detectable at any of the times tested (4, 12, 24 and 36 hr). The defect in TfR gene expression is selective since IL2Rα gene expression was induced in T‐TIL Because IL2 binding to its receptor results in TfR expression, the defect in TfR induction in T‐TIL appears to be distal to IL2Rα expression. Our studies illustrate another alteration in T‐TIL that is not observed in T cells from the peripheral blood. The absence of TfR gene expression may contribute to the poor proliferative response of T cells from the tumor.
Expression of transforming growth factor‐beta (TGF‐β) receptors, TGF‐β1 and TGF‐β2 production and autocrine growth control in osteosarcoma cells
Authors: Kloen Peter , Jennings Candace L. , Gebhardt Mark C. , Springfield Dempsey S. , Mankin Henry J.
Published: February 2007

Transforming growth factor‐beta (TGF‐β) is a polypeptide with multiple physiological functions. Isoforms of this growth factor have important roles in control of the cell cycle, in regulation of cell‐cell interactions and in growth and development. Malignant transformation has been shown to be associated with increased expression of TGF‐β. Since bone is the largest storage site and producer of TGF‐β, we speculated on the existence of an autocrine mechanism in osteosarcoma, a malignant bone tumor. Expression of TGF‐β cell surface receptors, effects on growth of TGF‐p and TGF‐β antibodies and production of 2 TGF‐β isoforms were studied in a panel of 7 osteosarcoma cell lines. In contrast to most previous reports on the effects of TGF‐β on osteosarcoma cell growth, we found a mitogenic effect of TGF‐β 1 in 4 of 7 osteosarcoma cell lines. Receptor profiles for TGF‐β were aberrant in 5 of the 7 cell lines tested, and production of TGF‐β1 and TGF‐β2 varied among cell lines. Addition of anti‐TGF‐β antagonized the effects of endogenous TGF‐β. Our results suggest a potential role of TGF‐β in autocrine growth control of osteosarcoma cells.
Regulation of intercellular adhesion molecule‐1 expression by retinoic acid: Analysis of the 5′ regulatory region of the gene
Authors: Aoudjit Fawzi , Bosse Marc , Stratowa Christian , Voraberger Günter , Audette Marie
Published: February 2007

Human intercellular adhesion molecule‐1 (ICAM‐1), a specific ligand for the lymphocyte‐function‐associated antigen‐1, plays an important role in immune responses. ICAM‐I expression is regulated by various proinflammatory cytokines, by PMA, and by retinoic acid. In this study, we have investigated the mechanisms of transcriptional control involved in the stimulation of ICAM‐I gene expression by retinoic acid in SK‐N‐SH cells. Northern‐blot analysis demonstrated that ICAM‐I mRNA is maximally induced at 24 hr, suggesting that it is not an early‐response gene with respect to retinoic‐acid responsiveness, whereas the retinoic acid receptorβ mRNA level was maximal 12 hr following retinoic acid treatment. To analyze the 5′‐regulatory region of the ICAM‐I gene, an EcoRI/Sall fragment spanning the first 1.3 kb upstream of the translational start site was used to direct the expression of a linked luciferase reporter gene in transient transaction assays in SK‐N‐SH cells. A 24‐hr treatment of transfected cells with 10μ retinoic acid resulted in a 10‐ to 13‐fold increase in luciferase activity compared with untreated cells. Deletion mutant analysis revealed that a region located between ‐393 and ‐ 176 bp from the translational start site is critical for retinoic acid stimulation of luciferase activity. This region harbors a consensus sequence for a retinoic‐acid‐responsive element (RARE) homologous to the element found upstream of the alcohol dehydrogenase‐3 gene. Co‐transfection of expression vectors encoding the retinoic acid receptor‐α, ‐β, or ‐γ, with reporter plasmids harboring the putative RARE, confirmed that the ICAM‐I gene is regulated by retinoic acid in a retinoic acid receptor‐dependent fashion.
The PRAD‐1/cyclin D1 oncogene product accumulates aberrantly in a subset of colorectal carcinomas
Authors: Bartkova Jirina , Lukas Jiri , Strauss Michael , Bartek Jiri
Published: February 2007

The PRAD‐l/cyclin Dl proto‐oncogene is localized on chromosome 11 q 13 and it is overexpressed in several tumour types as a consequence of gene amplification or chromosomal rearrangements. In this study, the abundance and patterns of cyclin Dl protein expression in normal/non‐involved colon (n = 44), primary (n = 48) and metastatic (n = 9) colorectal carcinomas, and in a series of 4 colon cancer cell lines were investigated by immunochemical methods using the DCS‐6 monoclonal antibody specific for cyclin Dl. While examination of all normal colorectal tissue samples and 56% of the primary tumours revealed only weak to undetectable immunostaining signals, 23% of the primary carcinomas showed moderate and 21% showed strong aberrant accumulation of this cell‐cycle regulatory oncoprotein. The immunohistochemical patterns in the secondary lesions were concordant with the matched primary tumours in all cases. The staining was nuclear both in the clinical specimens and in the colon cancer cell lines, in which the antibody‐mediated knock‐out experiments demonstrated a positive regulatory role of the cyclin Dl protein whose function was required for progression through the G1 phase of the cell cycle. These results indicate that the PRAD‐l/cyclin Dl protooncogene may be deregulated in a significant subset of colorectal tumours, and warrant further analyses of such aberrations of the cyclin Dl/retinoblastoma protein pathway to elucidate its potential involvement in the multistep pathogenesis of human colorectal cancer.
Sulfonated aluminium phthalocyanines as sensitizers for photochemotherapy. Effects of small light doses on localization, dye fluorescence and photosensitivity in V79 cells
Authors: Moan Johan , Berg Kristian , Anholt Helle , Madslien Kari
Published: February 2007

V79 cells incubated with di‐ or tetrasulfonated aluminium phthalocyanines (AIPcS2 or AIPcS4) showed a granular fluorescence pattern. Co‐staining with the lysosomotropic dye acridine orange (AO) indicated that the granules that were stained by these photoactive phthalocyanines were identical to lysosomes. Small light exposures made the lysosomes permeable to the dyes without inactivating the cells. Also, the lysosomal enzymes β‐AGA and cathepsin (L + B) were inactivated by small light exposures when AIPcS4 was present. Such small and almost nontoxic light exposures caused a redistribution of the dyes in the cells that was accompanied by a more than 10‐fold increase in the fluorescence quantum yields of the dyes. Surprisingly, this redistribution and increase in fluorescence did not result in any significant increase in the photosensitivity of the cells.
Fermented dairy products, dietary calcium and colon cancer: A case‐control study in the netherlands
Authors: Kampman Ellen , Van't Veer Pieter , Hiddink Gert Jan , Van Aken‐Schneijder Petra , Kok Frans J. , Hermus Rudolph J. J.
Published: February 2007

To examine whether the consumption of fermented dairy products or the dietary intake of calcium decreases colon cancer risk, a case‐control study was conducted in The Netherlands. Dietary patterns were assessed in detail (for cases before diagnosis or symptoms occurred) using a structured dietary history questionnaire. After adjustment for potential confounding variables, consumption of fermented dairy products, hard cheese and unfermented dairy products was not significantly associated with risk of colon cancer: an odds ratio (OR) of I. I was found for individuals consuming more than one serving of fermented dairy products per day as compared to those consuming less than 10% of one serving a day. Adjustment for dietary calcium attenuated the associations. Total dietary calcium was positively but non‐significantly associated with colon cancer risk after adjustment for age, gender, urbanization level and total energy intake. Additional adjustment for a positive family history of colorectal cancer, cholecystectomy and energy‐adjusted intake of total fat, dietary fibre, vitamin C and alcohol increased the association. No differences were observed between calcium from fermented and from unfermented dairy sources. The observed associations for fermented dairy products and dietary calcium differed between men and women: positive significant associations were observed in men, while in women non‐significant inverse associations were found. Our results do not support the hypothesis that an increased intake of commercially available, commonly used fermented dairy products or dietary calcium decreases the risk of colon cancer.
Production and response of human prostate cancer cell lines to granulocyte macrophage‐colony stimulating factor
Authors: Lang Shona H. , Miller William R. , Duncan William , Habib Fouad K.
Published: February 2007

Prostate cancer selectively metastasises to skeletal sites, where it normally produces osteoblastic lesions. This study investigated whether haematopoietic growth factors known to be present in the bone environment could be involved in the survival and proliferation of prostate skeletal metastases. To evaluate this hypothesis we investigated the effects of recombinant granulocyte/macrophage colony‐stimulating factor (rGMGSF), recombinant granulocyte colony‐stimulating factor (rGCSF), recombinant erythropoietin (rEPO) and recombinant interleukin‐3 (rIL‐3) on the growth of 3 human prostate cancer cell lines. Two hormone‐insensitive cell lines, PC‐3 and DUI45, were significantly stimulated by rGM‐CSF and rEPO in serumfree medium but their growth was unaffected by incubation with rIL‐3 or rG‐CSF. A hormone‐sensitive cell line, LNCaP, was stimulated only by rGM‐CSF.

To investigate further the involvement of GM‐CSF in prostate cancer, the presence of GM‐CSF protein in the 3 prostate cancer cell lines was examined by immunohistochemistry, and analysis of cell line conditioned media was carried out by ELISA and Western blotting. These techniques demonstrated that GM‐CSF‐like material was produced by both DUI45 and PC‐3 cells but not by LNCaP. The results from ELISA found that media conditioned by DUI45 and PC‐3 cells contained 1.7 and 2.5 pg GM‐CSF/μg protein, respectively, whereas no GM‐CSF was detectable in the LNCaP conditioned media. Our results were also confirmed by Western blot analysis demonstrating one single band for DUI45 and PC‐3 conditioned media which co‐migrated along with the standard rGM‐CSF band. No bands were associated with the LNCaP conditioned media. The presence of GM‐CSF gene transcripts in DUI45 and PC‐3 cells was established by reverse transcription and polymerase chain reaction of total RNA.
H‐2Kb AND H‐2Db gene transfections in B16 melanoma differently affect non‐immunological properties relevant to the metastatic process. Involvement of integrin molecules
Authors: De Giovanni Carla , Nicoletti Giordano , Sensi Marialuisa , Santoni Angela , Palmieri Gabriella , Landuzzi Lorena , Nanni Patrizia , Lollini Pier‐Luigi
Published: February 2007

Modification of non‐immunological cell adhesion properties plays a major role in the decrease in metastatic ability observed after transfection of the H‐2Kb gene in H‐2‐negative BI6‐derived melanoma clone cells. To investigate the role played by different class‐l major histocompatibility complex (MHC) genes on non‐immunological properties relevant for metastasis, transfection with the H‐2Db gene alone or in conjunction with the H‐2Kb gene was performed. H‐2Db gene transfection did not modify either metastatic potential or non‐immunological cell adhesion properties. Double KbDb transfectants showed a decreased metastatic ability when compared to control clones and to Db transfectants; a decrease in homotypic adhesive ability was also observed, even though not in all clones studied: therefore expression of the Db gene is also relevant. The mechanisms of homotypic cell adhesion were studied and found to be dependent upon temperature and divalent cations. Adhesion was partially inhibited by an antiserum directed against the β1 integrin subunit, whereas anti‐αllbβ3 was ineffective. Cell pre‐treatment with anti‐β1 serum reduced metastatic ability. A decreased expression of α4 and α6 integrin subunits was observed in Kb clones, whereas no difference in the levels of some homophilic cell adhesion molecules, such as N‐CAM and αllbβ3, was found. Adhesion required the activity of tyrosine kinases, as suggested by the decreased adhesive properties and impaired metastatic ability of cells pre‐treated with the tyrosine‐kinaseinhibitor genistein. These results are compatible with involvement of integrin molecules of the β1 family in the adhesive ability of these cells. Our data show that: (a) immunological and non‐immunological effects of MHC transfection are correlated and depend on the class‐l gene used, suggesting that MHC gene therapy can be highly successful only if appropriate MHC genes are transfected; (b) non‐immunological cell‐adhesion properties modified after MHC transfection could be related to an impairment of integrin‐mediated adhesive interactions.
Resistance to verapamil sensitization of multidrug‐resistant cells grown as multicellular spheroids
Authors: Sakata Koh‐Ichi , Kwok Tim T. , Gordon G. Ross , Waleh Nahid S. , Sutherland Robert M.
Published: February 2007

The ability of verapamil to overcome resistance to adriamycin in a multidrug‐resistant derivative of the V79 cell line (LZ), grown as multicellular spheroids or as monolayers, was examined. Verapamil was much less effective in overcoming resistance to adriamycin in spheroids than in monolayers. Verapamil increased the adriamycin content of cells grown as monolayers, but had no significant effect on the drug content of spheroids. This occurred in spite of the same mdr‐I mRNA and protein levels in monolayers and spheroids. When the surviving fraction of cells was normalized to the cellular adriamycin content, cells both in monolayers and spheroids treated with verapamil were still more sensitive to adriamycin than their counterparts not treated with verapamil. The observed resistance of spheroids to adriamycin and verapamil sensitization may be caused by a drug‐resistance mechanism that is functional only in spheroids, in addition to the activity of P‐glycoprotein. Multicellular tissue architecture and cell‐cell contact may play significant roles in this type of multidrug‐resistance mechanism.
Neuroblastoma cell apoptosis induced by the synthetic retinoid N‐(4‐hydroxyphenyl)retinamide
Authors: Di Vinci Angela , Geido Elio , Infusini Edmondo , Giaretti Walter
Published: February 2007

N‐(4‐hydroxyphenyl)retinamide (HPR) is a synthetic retinoid with anti‐cancer properties and lower toxicity than all‐trans retinoic acid (RA). Neuroblastoma cells treated with HPR and observed by fluorescence microscopy showed clear signs of apoptosis, such as chromatin condensation and margination, nuclear fragmentation and the presence of “apoptotic bodies”. Moreover, measurements on a cell‐by‐cell basis by the flowcytometric DNA‐content in situ‐terminal‐deoxinucleotidyltransferase(TDT) assay showed that apoptosis induced by HPR was dose‐ and time‐dependent and that the fraction of apoptotic cells increased from approximately 15% at 1.25 μM at 2 days after treatment up to approximately 90% at 5 μM and 8 days of continuous treatment. Additionally, we found that cells were induced into apoptosis independently from the cell‐cycle phase. In contrast, equimolar or higher doses of RA, from 5 μM to 80 μM, were able to inhibit growth by differentiation, but failed to induce apoptosis. We conclude that the functional effects of HPR and RA in LA‐N‐5 neuroblastoma cells are mediated by apoptosis and differentiation respectively, suggesting a potential clinical use of HPR in the management of neuroblastoma patients. © 1994 Wiley‐Liss, Inc.
The fibronectin isoform containing the ed‐b oncofetal domain: A marker of angiogenesis
Authors: Castellani Patrizia , Viale Giuseppe , Dorcaratto Alessandra , Nicolo Guido , Kaczmarek Janusz , Querze Germano , Zardi Luciano
Published: February 2007

Different fibronectin (FN) isoforms are generated by the alternative splicing of 3 regions (ED‐A, ED‐B and IIICS) of the primary transcript. The FN isoform containing the ED‐B sequence, a complete type‐III‐homology repeat, while having extremely restricted distribution in normal adult tissues, reveals high expression in fetal and tumor tissues. Using the monoclonal antibody (MAb) BC‐1, specific for the FN isoform containing the ED‐B sequence (B+·FN), we demonstrate here, using immunohistochemical techniques, that while this FN isoform is undetectable in mature vessels, it is highly expressed during angiogenesis both in neoplastic and in normal tissues, as in the case of the functional layer of endometrium during the proliferative phase. B+·FN is thus a marker for the formation of new vessels, and the BC‐I MAb may be a useful reagent for evaluating the level of the angiogenetic process in different neoplasms.
Protracted treatment with phorbol ester modulates j‐7 human hepatoma‐cell‐induced aggregation and coagulation of human platelet‐rich plasma
Authors: Ko Feng N. , Yu Sheu M. , Teng Che M.
Published: February 2007

In heparinized human platelet‐rich plasma (PRP), J‐7 human hepatoma cells initially induced platelet aggregation; then a clot formed. ADP‐scavenger systems, apyrase and creatine phosphate/creatine phosphokinase did not inhibit this tumor‐cell‐induced platelet aggregation (TCIPA), whereas hirudin and concanavalin A completely blocked it. J‐7 cells also shortened the recalcification time of normal and of Factor‐VIII‐ and ‐IX‐deficient human plasma, although it was inactive in shortening the recalcification time of Factor‐VII‐deficient plasma. After treatment with phorbol 12,13‐dibutyrate (PDBu) for 5 to 90 min, the aggregation and coagulation abilities of J‐7 cells were unaffected. Prolonged treatment of J‐7 cells with PDBu but not with α‐PDBu for 24 and 72 hr resulted in gradual loss of aggregation and coagulation. Staurosporine antagonized the effect of PDBu and restored aggregation and coagulation in J‐7 cells. Protracted treatment with PDBu (24 or 72 hr) did not affect adherence of J‐7 cells to the extracellular‐matrix proteins (i.e., fibrinogen, fibronectin, laminin, vitronectin and collagen types I and IV) or to the surface of plastic culture dishes. The treatment also did not affect J‐7 cell detachment from plastic culture dishes. These in vitro results demonstrate that protracted phorbol ester treatment diminishes TCIPA and blood coagulation of tumor cells.
Role of mature leukemic cells in the amplification of leukemic stem cells in a murine model
Authors: Dührsen Ulrich , Knieling Gabriele , Hossfeld Dieter Kurt
Published: February 2007

The recently described PGM‐2 leukemia displays a hierarchical structure with bipotential stem cells, B‐lymphocyte and macrophage progenitor cells, and post‐mitotic end cells. Because the different cell types can easily be identified in vitro by clonal culture assays and simple staining procedures, this leukemia is a useful model for the study of the interactions between different cell compartments in a leukemic clone. Our analysis of the impact of mature leukemic macrophages on the proliferation of stem cells was facilitated by the establishment of long‐term cultures producing new stem cells over prolonged periods of time. A prerequisite was the development of an adherent layer of fibroblasts and leukemic macrophages. Enumeration of adherent cells revealed a good correlation between the number of macrophages and the number of stem cells generated, and expansion of the macrophage population by treatment with interieukin 3 (IL‐3) resulted in a significant improvement of the culture conditions. Leukemic macrophages were also able to induce the formation of stem‐cell colonies in agar culture, suggesting a role for humoral mediators. Antibody neutralization experiments and bioassays identified IL‐7 and IL‐6 as factors cooperating in the stimulation of stem‐cell self‐renewal. Feed‐back stimulation of leukemic stem cells by mature leukemic cells may also be relevant to human leukemias and have implications for differentiation therapy.
Nucleotide sequence analysis of a full‐length human T‐cell leukemia virus type I from adult T‐cell leukemia cells: A prematurely terminated PX open reading frame II
Authors: S.Chou Kuan , Okayama Akihiko , Tachibana Nobuyoshi , Lee Tun‐Hou , Essex Max
Published: February 2007

Human T‐cell leukemia virus type 1 (HTLV‐I) is etiologically associated with adult T‐cell leukemia/lymphoma (ATL). The prototypic HTLV‐I, ATK, is the only full‐length provirus cloned from uncultured leukemic cells and completely sequenced prior to this study. We have determined the complete nucleotide sequence of another full‐length HTLV‐I provirus cloned directly from leukemic cells. A premature termination codon was found in the second open reading frame (orf II) of the pX region. Our finding indicates that open reading frame II of the HTLV‐I pX region is not required for outgrowth of ATL leukemic clones in vivo.
Modulation of intraepithelial expansion of human T24 bladder‐carcinoma cells in murine urothelium by growth factors and extracellular‐matrix components
Authors: Rebel Johanna M. J. , Thijssen Cornelia D. E. M. , Vermey Marcel , Zwarthoff Ellen C. , Van Der Kwast Theodorus H.
Published: February 2007

The high recurrence rate of bladder cancer is probably due to an efficient repopulation of the bladder by residual transformed cells after resection of the tumour. However, the regenerating capacity of the normal urothelial cells is very high. To study the balance between regenerating normal urothelium and outgrowth of transformed urothelial cells, we recently developed an in vitro co‐cultivation model. With this model system we studied the effects of growth factors and extracellular‐matrix components on the intra‐epithelial expansion of human T24 bladder‐carcinoma cells in primary mouse‐bladder explants. Exposure of the cultures to acidic fibroblast growth factor (aFGF) and laminin led to a dramatic increase in the number of invasive T24 cells into the primary urothelium. Epidermal growth factor (EGF) and collagen types I and IV counteracted the infiltration of individual T24 cells. EGF, aFGF, laminin and collagen types I and IV did not directly affect the migration and proliferation of T24 cells. Apparently, the efficacy of invasion of transformed urothelial cells into primary urothelium is not only dependent on the intrinsic characteristics of the transformed cells, but can be influenced to a considerable extent by exogenous components exerting their influence on the normal urothelium. The clinical relevance of this observation needs to be studied further.
Targeted inhibition of tumor‐cell growth by recombinant heregulin‐toxin fusion proteins
Authors: Jeschke Margit , Wels Winfried , Dengler Wolfgang , Imber Roland , Stöcklin Elisabeth , Groner Bernd
Published: February 2007

Fusion of functional domains of proteins by in vitro recombination of gene fragments can be used to generate novel anti‐tumor agents. The combination of tumor‐cell‐recognition functions and toxic functions results in cytotoxic molecules with a high specificity for tumor cells. Human adenocarcinomas are frequently characterized by over‐expression of members of the epidermal‐growth‐factor (EGF) receptor family (ErbB‐1, 2, 3 and 4), when compared with normal cells. These tumors are particularly suited to treatment with recombinant toxins. The human heregulins (HRG) and their rat counterparts (neu differentiation factor, NDF) have been identified as ligands for these receptors. Two chimeric heregulin‐toxin fusions consisting of the EGF‐like receptor recognition domain of the heregulin isoforms HRGα and HRGβ1, and the domains II, lb and III of the Pseudomonas exotoxin A (ETA) were constructed. HRGβ1‐ETA is highly cytotoxic for the mammary carcinoma cell lines SK‐BR‐3 and MDA‐MB‐453. HRGα‐ETA was less active than HRGβ1‐ETA. The killing activity of the recombinant toxins correlated with the expression levels of ErbB‐3 and/or ErbB‐4 in the cell lines studied. High expression of ErbB‐2 is not sufficient to confer sensitivity towards the HRG‐ETA. Treatment of mice with 0.4 mg/kg/day of HRGβ1‐ETA caused growth retardation of transplanted human breast tumor cells. Higher levels of HRGβ1‐ETA administration resulted in acute hemorrhagic necrosis of the liver.
Molecular detection of pre‐atl state among healthy HTLV‐1 carriers in an endemic area of Japan
Authors: Ghen Yun‐Xian , Jkeda Shuichi , Mori Hiroyuki , Hata Tomoko , Tsukasaki Kunihiro , Momita Saburo , Yamada Yasuaki , Kamihira Shimeru , Mine Mariko , Tomonaga Masao
Published: February 2007

Adult T‐cell leukemia/lymphoma (ATL) usually develops after age 40 in Japan, suggesting a long latency since HTLV‐I is considered to be transmitted mainly from mothers to babies via breast milk. Our previous studies had suggested that HTLV‐I carriers who have a monoclonal integration of HTLV‐I proviral DNA in the peripheral blood cells, designated pre‐ATL, account for about 2% of healthy carriers and are at high risk of developing ATL Their ages ranged from 32 to 80 (median 57). Nevertheless, many cases of pre‐ATL showed a long‐lasting carrier state (10‐year probability around 90%). In the present investigation we conducted a large‐scale molecular detection of monoclonal integration in a population (481 cases) of healthy carriers with ages ranging from 16 to 82 (median 49) for the purpose of clarifying the earliest onset of this pre‐ATL state. Southern‐blot analysis of DNA extracted from peripheral blood mononuclear cells revealed 6 cases (1.2%) with a monoclonal band. All of these 6 were older than 40; no single positive case was found in 220 carriers under age 40. These results indicate that the molecularly detectable pre‐ATL state also develops after a long latency. Thus the pre‐ATL state seems to be a subtype of ATL showing an extremely indolent course of disease development, but not merely an early phase of all subtypes of ATL. We propose that, in order to reveal a promoter(s) responsible for the development of ATL from the pre‐ATL state, intensive epidemiological investigation of life style, eating habits, occupation and exposure to carcinogens must be conducted on this unique group prone to ATL. It is also important to perform such an epidemiological study on the general population of carriers by paying special attention to the first 3‐4 decades of each carrier's life in Japan and by comparing the data with those from carriers in different geographical areas of the world.
Inhibition of invasion, gelatinase activity, tumor take and metastasis of malignant cells by N‐acetylcysteine
Authors: Albini Adriana , D'Agostini Francesco , Giunciuglio Daniela , Paglieri Isabella , Balansky Roumen , De Flora Silvio
Published: February 2007

The thiol N‐acetylcysteine (NAC) is currently considered one of the most promising cancer chemopreventive agents by virtue of its multiple and coordinated mechanisms affecting the process of chemical carcinogenesis. Recent studies have shown that an unpaired cysteine residue in the propeptide plays a key role in inactivation of latent metastasis‐associated metalloproteinases: the present study was designed to assess whether NAC could also affect tumor take, invasion and metastasis of malignant cells. As assessed by zymographic analysis, NAC completely inhibited the gelatinolytic activity of type‐IV collagenases in the cells tested (gelatinases A and B). Moreover, NAC was efficient in inhibiting the chemotactic and invasive activities of tumor cells of human (A2058 melanoma) and murine origin (K1735 and B16‐F10 melanoma cells as well as C87 Lewis lung carcinoma cells) in Boyden‐chamber assays, which are predictive of the invasive and metastatic properties. Reduced glutathione (GSH) had a similar, although less effective activity. The number of lung metastases decreased sharply when B16‐F10 murine melanoma cells, injected i.v. into nude mice, were pre‐treated with NAC and resuspended in medium supplemented with 10 mM NAC. In other experiments NAC was given in drinking water, starting 48‐72 hr before subcutaneous inoculation of either B16‐F10 cells or of their highly metastatic variant B16‐BL6, or intramuscular injection of LLC cells. In all experiments NAC treatment decreased the weight of the locally formed primary tumor and produced a dose‐related delay in tumor formation. Spontaneous metastasis formation by B16‐F10 and B16‐BL6 tumors was slightly yet significantly reduced by oral administration of NAC. However, this was not observed for Lewis lung tumors. These data indicate that NAC affects the process of tumor‐cell invasion and metastasis, probably due to inhibition of gelatinases by its sulfhydryl group, with the possible contribution of other mechanisms, including the potent antioxidant activity of this thiol. © 1995 Wiley‐Liss, Inc.
Suppression of pulmonary metastasis by angiogenesis inhibitor TNP‐470 in murine osteosarcoma
Authors: Mori Shigeki , Ueda Takafumi , Kuratsu Shigeyuki , Hosono Noboru , Izawa Kazutaka , Uchida Atsumasa
Published: February 2007

We treated a murine osteosarcoma cell line, LM8, which preferentially metastasizes to the lungs, with a new angiogenesis inhibitor, TNP‐470, to evaluate the efficacy of this compound in the suppression of pulmonary metastasis of osteosarcoma. In an in vivo experiment, tumor cells were inoculated i.v. into C3H mice, and TNP‐470 or vehicle alone (control group) was administered s.c. every day for 3 weeks. In the TNP‐470‐treated groups, both the number of pulmonary metastatic nodules and the lung wet weight were significantly reduced in a dose‐dependent manner. Similarly, vascular density in the metastatic tumors estimated by immunohistochemical staining with anti‐von‐Willebrand factor antibody as an endothelial marker were significantly reduced. No severe side‐effects were found. In an in vitro experiment, viable tumor cells were counted after 3 days' treatment with TNP‐470. The 50% inhibitory concentration was 0.6 ng/ml for LM8, which was more sensitive than other tumor cells previously reported. Our results show that TNP‐470 suppresses the pulmonary metastasis of LM8 and suggest that both its anti‐angiogenic activity and cytostatic activity towards LM8 are responsible for the anti‐tumor effect. © 1995 Wiley‐Liss, Inc.
Perinatal characteristics and adult mammographic patterns
Authors: Ekbom Anders , Thurfjell Erik , Hsieh Chung‐Cheng , Trichopoulos Dimitrios , Adami Hans‐Olov
Published: February 2007

We retrieved breast mammograms for 370 women 40 to 74 years old with no history of breast cancer, for whom birth weight, birth length, placental weight and other birth characteristics were indicated in their standard birth records at the Uppsala University Hospital. Blind evaluation of the mammograms allowed these to be classified according to Wolfe's pattern. Logistic regression analysis was applied using as independent variables the recorded birth characteristics and as outcome variable, high risk (P2 and DY) versus low risk (N1 and P1) mammographic parenchymal pattern. After controlling for all the recorded variables, the odds ratio for a high‐risk pattern (P2 or DY) increased consistently and significantly (P for trend 0.02) with the weight of the placenta, i.e. the main estrogen‐producing organ during pregnancy. There were weak and non‐significant positive associations with increasing birth weight (P for trend 0.53) and birth length (P for trend 0.52). These results are compatible with hypotheses suggesting that pregnancy estrogens or other perinatal characteristics may play a risk‐modulating role influencing breast cancer in the offspring. © 1995 Wiley‐Liss, Inc.