bluuebunny/crossref_metadata_2025 huggingface dataset:
Transformed rat pancreatic islet‐cell lines established by BK virus infection in vitro
Authors: Haukland Hanne Husom , Vonen Barthold , Traavik Terje
Published: February 2007

Single‐cell suspensions of primary rat pancreatic islet cells were infected with BK virus (BKV) prototype (Gardner) and the naturally occurring BKV strain (TU), isolated from human urine. These viral strains have different sequences in their non‐coding control regions which contain promoter‐enhancer elements and origin of DNA replication. Uninfected cell cultures disintegrated within 3 weeks, while a varying number of virus‐infected cultures were immortalized and a majority exhibited focal areas of 2‐dimensional growth. A significantly higher proportion of the BKV (TU)‐ than of the BKV (Gardner)‐infected cultures were immortalized. Large tumor (T) antigen expression was evident in virus‐infected cultures from day 4 post infection (p.i.), while insulin secretion decreased steadily during the first weeks of culture. Two cell lines were established from BKV (TU)‐infected cultures. Line 5A4 was contact‐inhibited, growing as a dense monolayer, while 6A3 demonstrated foci of 2‐dimensional growth. Both cell lines have retained their morphology and T‐antigen expression for approximately 130 passages. At high passages a low level of intracellular insulin, but no secretion into media, was detected. Based on standard biological tests (reduced serum requirements, growth at low cell density, anchorage‐independent growth and tumor induction in nude mice) both cell lines have a fully transformed phenotype, but 6A3 appears to be more malignantly transformed. Since both cell lines were established simultaneously from the same primary cells with the same virus batch, they provide an opportunity to study the transforming mechanisms of BKV in a relative context.
Some approaches to improve the therapeutic effectiveness of adoptive chemoimmunotherapy with spleen cells from melphalan‐treated BALB/C mice bearing a large MOPC‐315 tumor
Authors: Weiskirch Larry M. , Mokyr Margalit B.
Published: February 2007

Spleen cells from BALB/c mice that are in the process of eradicating a large MOPC‐315 tumor following low‐dose L‐PAM therapy (L‐PAM TuB spleen cells) were previously shown to be capable of bringing about the complete regression of a large (15 to 20 mm) s.c. MOPC‐315 tumor in a substantial percentage of T‐cell‐deficient (athymic nude) mice that had been treated with low‐dose L‐PAM (adoptive chemo‐immunotherapy; ACIT). Here we show that aggressive depletion of CD4+ T‐cells by treatment both of spleen‐cell donors and of recipients with anti‐L3T4 monoclonal antibody (MAb) greatly improved the therapeutic effectiveness of L‐PAM TuB spleen cells in ACIT. In the light of the apparent importance of cytotoxic T‐lymphocytes (CTLs) for tumor eradication in low‐dose L‐PAM‐treated MOPC‐315‐tumor bearers, it is interesting that treatment of L‐PAM TuB spleen‐cell donors with anti‐L3T4 MAb was found to result in the generation of enhanced splenic anti‐MOPC‐315 cytotoxicity. Although most athymic nude mice in which the tumor had apparently completely regressed following ACIT remained tumor‐free, approximately 1/3 of the mice relapsed. However, a substantial percentage of the relapsing mice were rescued by a low dose of L‐PAM, which was not effective in causing tumor regression in athymic nude mice bearing a comparably large tumor if the mice had not been subjected previously to ACIT. Almost all athymic nude mice that had been „cured”︁ of a large MOPC‐315 tumor by ACIT but did not resist a subsequent MOPC‐315 tumor challenge were rescued by low dose L‐PAM. Thus, the therapeutic effectiveness of L‐PAM TuB spleen cells in ACIT may be improved by aggressive depletion of CD4+ T‐cells, suggesting that a low dose of L‐PAM, which leads to the acquisition of potent splenic‐tumor‐eradicating immunity in BALB/c mice bearing a large MOPC‐315 tumor, does not eliminate completely (or possibly not at all) the inhibitory activity of CD4+ T‐cells. In addition, athymic nude mice that are not endowed with fully protective tumor‐eradicating immunity following ACIT still have a substantial residual anti‐tumor immune potential that can be exploited to bring about eradication of a large tumor burden following low‐dose L‐PAM therapy.
An organoid culture assay (OCA) for determining the drug sensitivity of human tumors
Authors: Köpf‐Maier P. , Kolon B.
Published: February 2007

A model for testing chemotherapeutic agents in vitro is described. It is based on an organoid culture method which allows human carcinomas to grow in vitro and to maintain many typical in vivo properties, including 3‐dimensional architecture, growth of multiple cell types, expression of morphological differentiation and formation of histotypical structures. The preservation of drug sensitivity and resistance under the conditions of our organoid culture assay (OCA) was demonstrated by investigating 3 strains of a human hypopharynx carcinoma which differed by different sensitivity to cisplatin in in vivo conditions. These differences were retained in vitro and the modified neutral‐red (NR) assay was especially suitable for revealing drug‐induced cytotoxic damage in OCA. On the basis of our findings, the following approach is proposed for the in vitro testing of cytostatic drugs before they are administered to patients. (1) Removal of carcinomas from patients; (2) dense cell suspensions of these carcinomas to be dropped on membrane filters at the air‐medium interface, resulting in growth of solid nodules of organized carcinoma tissues; (3) addition of cytostatic drugs to the growth medium for 2 or 3 days; (4) detachment and bisection of the culture nodules; (5) determination of viable cells by NR uptake and total cell mass by the sulforhodamin B (SRB) assay; (6) determination of quotient NR:SRB absorbance, related as percentage to control value: this indicates the fraction of viable cells and gives a measure of the cytotoxic injury caused by the applied cytotoxic drug. Thus, the OCA seems to be suitable for defining the patterns of drug sensitivity and resistance of individual human carcinomas in vitro within a few days.
The use of molecular genetic markers to demonstrate the effect of organ environment on clonal dominance in a human renal‐cell carcinoma grown in nude mice
Authors: Staroselsky Alexander N. , Radinsky Robert , Fidler Isaiah J. , Pathak Sen , Chernajovsky Yuti , Frost Philip
Published: February 2007

Transduction of the SNI2C human renal‐cell carcinoma line with the neoR gene produces a genetically „tagged”︁ cell population within which individual clones can be identified if they dominate the tumor during its growth in vivo. We used this technique to determine whether the clones that dominate the primary local tumor and its metastases are the same or different when the tumor is growing in different organ sites in nude mice. The results show that clonal dominance is influenced by the organ environment in which the primary tumor grows, i.e., distinct clones dominated in the kidney, colon and subcutaneous sites. In addition, tumors grown in the orthotopic site (kidney) were all populated by the same dominant clones, and each distant visceral metastasis retained the same clonality. SNI2C neoR‐cells grown in an epithelial, ectopic site (colon) produced tumors with uniquely different dominant clones, and their visceral metastases retained the dominant pattern expressed by the parent tumor from which they were derived. In contrast, SNI2C tumors growing subcutaneously showed a random pattern of clonal dominance in both their primary and metastatic sites. Parallel cytogenetic analyses could not demonstrate these patterns. We conclude that the organ environment significantly influences clonal dominance of human renal carcinoma and that tumor injection into orthotopic sites may produce a more reproducible selection of dominant clones.
Modulation by staurosporine of phorbol‐ester‐induced effects on growth and protein kinase C localization in a549 human lung‐carcinoma cells
Authors: Bradshaw Tracey D. , Gescher Andreas , Pettit George R.
Published: February 2007

12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and bryostatin I are activators of protein kinase C (PKC). TPA is a potent inhibitor of the growth of A549 cells, while bryostatin I exerts a weak antiproliferative effect upon this cell line. We tested the hypothesis that the PKC inhibitor staurosporine (STAU) can interfere with the effects of TPA or bryostatin I on A549 cells. STAU alone arrested A549 cell growth effectively with an IC50 of 0.65 nM as determined by cell counting after incubation for 96 hr. It also caused the release of lactate dehydrogenase from cells with an IC50 of 18.4 nM. On incubation with cells for up to 8 hr, STAU (100 nM) alone did not reduce thymidine incorporation into cells. However, it partially abrogated the inhibition of DNA synthesis caused by TPA or bryostatin 1(10 nM). The IC50 for inhibition by STAU of the activity of PKC purified from A549 cells was 6.1 nM. Localization and levels of PKC were studied by Western blot and phorbol ester receptor binding analyses. STAU (100 nM) did not prevent the TPA‐induced rapid redistribution of PKC to the cell membrane, but instead increased it by 25%. The PKC downregulation caused by TPA was not reduced in the presence of STAU. The results suggest that (i) PKC activation is involved in growth inhibition caused by TPA or bryostatin I in A549 cells, and (ii) subcellular localization or levels of PKC can be pharmacologically manipulated even under conditions of inhibited kinase function.
Presence of IGF‐I In human midgut carcinoid tumours—an autocrine regulator of carcinoid tumour growth?
Authors: Nilsson Ola , Wängberg Bo , Theodorsson Elvar , Skottner Anna , Ahlman Håkan
Published: February 2007

The presence of IGF‐I and IGF‐I receptors in human midgut carcinoid tumours has been investigated. Using immunocytochemistry, IGF‐I‐positive tumour cells were demonstrated in 11/11 tumour cases studied. Labelling of consecutive sections with antibodies against IGF‐I and proliferating cell nuclear antigen (PCNA)/cyclin demonstrated a co‐distribution of the 2 antigens in carcinoid tumours. Extracts of tumour tissues were subjected to radioimmunoassay and shown to contain significant amounts of IGF‐I. Reverse‐phase HPLC of tumour extracts demonstrated a major IGF‐I‐immunoreactive component eluting in the position of rhlGF‐I, but also 2 other more hydrophobic forms. Conditioned serum‐free media from primary cultures of carcinoid tumors contained detectable amounts of IGF‐I, indicating a spontaneous release of IGF‐I from tumour cells into the culture medium. Levels of IGF‐I in media were reduced (19%) after incubation of cultures with a somatostatin analogue for 4 days. IGF‐I receptors were observed on tumour cells in 4/10 tumours by immunocytochemistry. Tumour cells with immunoreactive IGF‐I receptors could be stimulated to enhanced growth, measured as an increase in DNA contents, by exogenous administration of IGF‐I every 3–4 days for 2 weeks. The results show that cultured human midgut carcinoid tumours secrete IGF‐I and that some of the tumours also have IGF‐I receptors. We therefore suggest that IGF‐I may act as an autocrine or paracrine regulator of carcinoid tumour‐cell growth.
MHC‐restricted responses of CD8+ AND CD4+ T‐cell clones from regional lymph nodes of melanoma patients
Authors: Chen Qiyuan , Hersey Peter
Published: February 2007

Regional lymph‐node (LN) lymphocytes may constitute an important defence against the spread of human melanoma beyond regional LNs. The present study was directed to clonal analysis of lymphocytes cultured either directly from the LNs or after stimulation in cultures with autologous melanoma (MLTC). T‐cell clones derived from MLTC reactions had either CD4+ or CD8+ phenotypes. Inhibition studies with monoclonal antibodies (MAbs) suggested that the CD8+ cytotoxic T‐cell (CTL) clones had MHC‐class‐I‐restricted cytotoxic activity against the autologous and a proportion of HLA‐class‐I‐compatible allogeneic melanomas. The pattern of cytotoxicity against a panel of HLA‐typed melanoma cells and inhibition by (polyclonal) HLA‐typing sera suggested the CD8+ CTL were restricted by HLA‐A3. The CD4+ T‐cell clones had weak cytotoxic activity which appeared restricted by HLA‐DR2. T cells cultured from unstimulated lymphocytes were all CD4+. One of the clones exhibited cytotoxic activity against both the autologous and HLA‐DR2‐compatible allogeneic melanoma cells, whereas another 2 had cytotoxic activity only against a HLA‐DR2‐compatible allogeneic melanoma established from a primary melanoma. IL‐2 production by a 4th non‐cytotoxic clone had similar specificity. These results suggest that HLA‐A3 and DR2 may act as restricting elements in recognition of melanoma antigens by T cells from LNs and that they may have recognized at least 2 different antigens on the melanoma cells.
Glycolipids carrying Ley are preferentially expressed on small‐cell lung cancer cells as detected by the monoclonal antibody MLuC1
Authors: Leoni Flavio , Colnaghi Maria I. , Canevari Silvana , Ménard Sylvie , Colzani Elsa , Facheris Patrizia , Figini Mariangela , Miotti Silvia , Magnani John L.
Published: February 2007

The monoclonal antibody MLuCI, which reacts strongly with a high percentage of small‐cell lung cancers (SCLC), as well as with various human carcinomas, has been used to immunochemically characterize the recognized epitope (CaMLuCI). To this aim 3 different approaches were adopted: (I) immunoblotting/ immunostaining of extracts from various tumor‐cell lines; (2) inhibition of binding by purified oligosaccharides; (3) direct binding to oligosaccharide‐protein conjugates. All of these experiments indicate that CaMLuCI is present on the Ley blood‐group structure heterogeneously expressed on various glycoproteins and glycolipids. The expression of the glycoconjugates carrying Ley was then analyzed on breast and lung cancers and on their normal counterparts. Our overall results suggest that SCLC produce Ley‐active glycolipids in higher amounts compared to other tumors of the same or of a different oncotype, as well as normal lung cells, thus indicating an SCLC‐specific modification of the glycosylation pathways.
Targeting liver tumors by administering liposomal doxorubicin into the hepatic artery
Authors: Zou Yiyu , Horikoshi Isamu , Ueno Masaharu , Gu Xueqiu , Perez‐Soler Roman
Published: February 2007

The plasma levels, organ distribution, and in vivo anti‐tumor activity of liposomal doxorubicin administered i.v. or i.a. (hepatic) in rats bearing W256 liver tumors were studied. I.a. administration of liposomal doxorubicin resulted in 4‐fold and 1.3‐fold higher liver tumor and liver parenchyma doxorubicin levels, respectively, than i.v. administration, thus suggesting a more preferential distribution of liposomal doxorubicin into the liver tumor with i.a. administration. By contrast, the heart, spleen, and plasma AUCs were decreased 3.8‐, 3.2‐, and 16‐fold, respectively, after i.a. administration. Cumulative urinary excretion at 8 hr was also 14 times lower in animals that received liposomal doxorubicin i.a. In good correlation with these findings, i.a. administration markedly enhanced the anti‐tumor effect of liposomal doxorubicin against liver W256 tumors as measured by tumor growth inhibition 5 days after treatment (‐16% for i.a. administration vs. +89% for i.v. administration, p ⩽ 0.05) and prolongation of survival (ILS: 108% for i.a. administration vs. 26% for i.v. administration, p ⩽ 0.05). Our results show that i.a. administration of liposomal doxorubicin results in preferential distribution of the anti‐tumor agent into the tumor tissue and increased anti‐tumor activity, while increasing the cardioprotective effect of the liposome carrier by decreasing the plasma peak and heart‐tissue levels of the drug.
Alternative splicing of neural‐cell‐adhesion molecule mRNA in human small‐cell lung‐cancer cell line H69
Authors: Moolenaar C. E. C. , Pieneman Cynthia , Walsh Frank S. , Mooi Wolter J. , Michalides Rob J. A. M.
Published: February 2007

The neural‐cell‐adhesion molecule (NCAM) is expressed in all small‐cell lung cancers (SCLC) and in approximately 20% of non‐small‐cell lung tumors (non‐SCLC). These NCAM‐positive lung tumors have a poor prognosis compared with NCAM‐negative tumors. Multiple NCAM protein isoforms are expressed from a single‐copy gene as a result of alternative splicing and/or post‐translational modifications. Therefore, we studied the NCAM isoforms expressed in a human small‐cell lung‐cancer cell line, H69. NCAM mRNA transcripts of 7.2, 6.7, 4.3 and 4.0 were detected in these cells on Northern blots. Since the various NCAM isoforms may have different biological properties, we performed a more precise examination of NCAM mRNAs, using polymerase chain reactions (PCR) with primers flanking the various NCAM exon boundaries. The shortest alternatively spliced sequence that we found was the trinucleotide AAG located between exon 12 and 13 in the so‐called hinge region of the NCAM protein. This AAG trinucleotide was present in the majority of the NCAM mRNAs. A second alternatively spliced 30 nt‐exon VASE (immunoglobulinvariable domain‐like alternatively spliced exon) was present in all NCAM transcript isoforms at the exon 7/exon 8 junction. VASE resulted in the insertion of 10 amino acids into the 4th immunoglobulin‐like loop of the NCAM protein. Within the limits of the PCR methodology, no evidence for the presence of mRNA containing exon 15, encoding the glycosyl‐phosphatidylinositol‐linked (GPI‐linked) NCAM isoform in H69 cells was obtained. Considering that H69 cells express 2 major NCAM protein classes (NCAM‐180 and NCAM‐140), and that the VASE and AAG alternative mRNA splice variants result in minor differences in protein sizes, at least 8 polypeptide isoforms of NCAM might be expressed in H69 cells that contribute to the binding interactions of NCAM.
In vivo anti‐tumor activity of arginine deiminase purified from Mycoplasma arginini
Authors: Takaku Haruo , Takase Megumi , Abe Shin‐Ichiro , Hayashi Hideya , Miyazaki Kaoru
Published: February 2007

Arginine deiminase (EC 3.5.3.6) was purified to homogeneity from the cell extract of Mycoplasma arginini by molecular‐sieve, anion‐exchange and arginine‐affinity chromatographies. The purified enzyme was composed of 2 identical sub‐units with a molecular weight of 45.000 and had a pl of 4.7. Its Vmax value and Km value for L‐arginine were estimated to be 50 units/mg protein and 0.2 mM, respectively. It exerted maximal enzyme activity at pH 6.0–7.5 and at 50°C. The arginine deiminase was stable at neutral pH. When injected i.v. into mice, the half‐life of the arginine deiminase in blood was about 4 hr. In culture, the enzyme strongly inhibited the growth of 6 kinds of mouse tumor cell lines by depleting L‐arginine in the culture media. When the in vivo growth‐inhibitory activity of arginine deiminase was tested for the 6 tumor cell lines, i.p. administration of the purified enzyme effectively prolonged the survival time of the mice injected with all kinds of the tumor cell lines. Especially, the in vivo growth of a hepatoma cell line, MH134, was completely prevented by the daily administration at a dose of 0.2 mg/mouse for 14 days. These results raise the possibility of the use of the arginine deiminase derived from Mycoplasma arginini as a new anti‐tumor drug.
ANti‐CD3 antibody‐treated mice: In Vivo induction of cytolytic activity and tnf production by lung leukocytes
Authors: Stanňková Jana
Published: February 2007

Anti‐CD3 antibody induced non‐MHC‐restricted cytolytic activity in murine spleen cells within 4 hr of incubation in vitro and within 8 hr in vivo after intraperitoneal injection. Interstitial lung leukocytes acquired the capacity to kill both NK‐sensitive and ‐resistant targets within 24 hr after anti‐CD3 injection. In vivo‐stimulated spleen cells produced significantly more IL‐2 and TNF than unstimulated cells. When these cells were restimulated with anti‐CD3 in vitro, a potentiation of TNF production was seen. Interstitial lung leukocytes doubled their TNF production ex vivo after anti‐CD3 injection, and the suppressed TNF production which was seen in lung cells from tumor‐bearing animals was restored after anti‐CD3 stimulation. The TNF production by alveolar macrophages was augmented 5‐fold 48 hr after injection of antibody. Anti‐CD3 antibody also induced significant accumulation of both TNFα and TNFβ mRNA in spleen and lung leukocytes. Pre‐treatment with anti‐TNF antibodies, both in vitro and in vivo, did not eliminate the cytolytic activation of lung and spleen cells. Our data indicate that anti‐CD3 antibodies can induce rapid activation of both cytolytic activity and cytokine production in lung lymphocytes and macrophages.
Immunity to B16 melanoma in mice immunized with IL‐2‐secreting allogeneic mouse fibroblasts expressing melanoma‐associated antigens
Authors: Kim Tae Sung , Russell Stephen J. , Collins Mary K. L. , Cohen Edward P.
Published: February 2007

Co‐presentation of weak tumour‐associated antigens along with strongly immunogenic determinants leads to the development of an anti‐tumour immune response in recipients syngeneic with the tumour. Tumour immunity develops in mice immunized with tumour cells modified by the introduction of cDNA for interleukin‐2 (IL‐2). Here, we report the anti‐tumour response following immunization with an IL‐2‐secreting cell construct that expresses tumour‐associated antigens, along with allogeneic major histocompatibility antigens. The construct was prepared by transfecting LM(TK−) mouse fibroblasts (H‐2k) with genomic DNA from B16 melanoma cells syngeneic in C57BL/6J mice (H‐2b). Transfectants expressing melanoma‐associated antigens (MAA) were then infected with an expression‐competent retroviral vector containing a cDNA specifying human IL‐2. Cytotoxicity toward B16 cells was detected for as long as 5 months in both spleen and macrophage cell populations in C57BL/6J mice immunized with the IL‐2‐secreting, cells. Mice immunized with non‐IL‐2‐secreting, MAA‐positive allogeneic cells developed melanoma immunity as well, but to a lesser extent. Immunity to 2 tumour‐cell lines expressing the H‐2d haplotype and to YAC‐I cells was detected in peritoneal macrophages, but not in spleen cells from C57BL/6J mice immunized with the cell construct, indicating that the response to B16 cells was only partially specific. C57BL/6J mice immunized with the IL‐2‐secreting cell construct survived significantly longer, following an injection of viable B16 cells, than mice in various control groups. The contribution of allogeneic antigens to the melanoma immunity was indicated by the failure of mice syngeneic with LM(TK−) cells to develop melanoma immunity following immunization with non‐IL‐2‐secreting, MAA‐positive cell constructs. The formation of IL‐2 partially compensated for the lack of allogeneic antigens.
Hematopoietic growth factors secreted by seven human pleural mesothelioma cell lines: Interleukin‐6 production as a common feature
Authors: Schmitter Doris , Lauber Béatrice , Fagg Barbara , Stahel Rolf A.
Published: February 2007

Seven mesothelioma cell lines, established from patients with pleural mesothelioma, exhibited substantial heterogeneity regarding in vitro morphology and growth characteristics. Media conditioned by these cell lines and by MeT5A normal mesothelial cells were examined for (i) colony formation on human bone‐marrow cells, (ii) hematopoietic growth‐factor content and (iii) mitogenic activity on mesothelioma cells. Colony‐stimulating activity was produced only by the ZL34 cell line. Analysis of conditioned media by ELISA revealed that all mesothelioma cell lines constitutively produced IL‐6, while the MeT5A normal mesothelial cells did not; in addition, GM‐CSF and G‐CSF were detected in the supernatant of the ZL34 cell line. Using a 3H‐thymidine incorporation assay, we showed that all mesothelioma cell lines produced mitogenic activity in the culture supernatant, in contrast to the MeT5A normal mesothelial cells. The mitogenic effect of the hematopoietic growth factors detected in mesothelioma culture supernatants was tested on mesothelioma cells and on MeT5A normal mesothelial cells: IL‐6, GM‐CSF and G‐CSF did not stimulate any DNA synthesis. Our results suggest that these hematopoietic growth factors do not act as autocrine growth factors. A common feature of this panel of mesothelioma cell lines is the production of IL‐6; although the biological significance of the aberrant production of cytokines by mesotheliomas remains unclear, IL‐6 might be involved in paraneoplastic syndromes such as thrombocytosis.
Pharmacokinetics and antitumor activity of epirubicin encapsulated in long‐circulating liposomes incorporating a polyethylene glycol‐derivatized phospholipid
Authors: Mayhew E. G. , Lasic D. , Babbar S. , Martin F. J.
Published: February 2007

The antitumor activity of epirubicin (EPI) entrapped in long circulating „Stealth”︁ liposomes containing a polyethylene glycolderivatized phospholipid (S‐EPI) was compared to epirubicin encapsulated in a conventional liposome formulation (L‐EPI) and free epirubicin (F‐EPI) against mouse colon 26 tumor in vivo. Pharmacokinetics of S‐EPI and F‐EPI were also compared in rats. F‐EPI was distributed to tissues within minutes of injection. In contrast, when administered in the S‐EPI formulation, the distribution half‐life of the drug was over 22 hr. S‐EPI also exhibited a reduced clearance compared to F‐EPI, from I I I to < 1.0 ml/hr. S‐EPI inhibited tumor growth more effectively than F‐EPI or L‐EPI by causing tumors to regress and increasing survival of mice. There were 9/10 (S‐EPI) compared to 0/10 (F‐EPI) 120‐day survivors when treatment was started 3 days after tumor implant. When treatment was delayed for 10 days, tumors, which had reached approx. 0.1–0.3 cm3 in volume, regressed in 8/10 animals receiving S‐EPI, whereas in all animals treated with F‐EPI the tumors progressed. L‐EPI was no more effective therapeutically than F‐EPI in this model. The maximum tolerated dose of S‐EPI was higher than that of F‐EPI. The enhanced therapeutic efficacy of S‐EPI is probably related to the extended circulation time of the formulation and its accumulation in tumors.
Harderian gland hyperplasia in c‐mos transgenic mice
Authors: Heath L. A. , Rosenberg M. P. , Thorogood P. , Speight P. , Propst F.
Published: February 2007

Transgenic mice carrying the mouse mos proto‐oncogene linked to a retroviral LTR develop hyperplasia of the Harderian glands. Enlargement of the glands is evident as early as 18 weeks after birth, with glands reaching up to 10 times their normal weight. Approximately 65% of the cases of hyperplasia occur bilaterally, and the majority of mice affected are male (66%). Elevated levels of mos expression are found in all Harderian glands of mice from the affected transgenic line, but not in glands of normal mice or a non‐affected transgenic line, indicating that hyperplasia is dependent on mos expression. Histological examination of the tissue reveals a general involvement of the entire gland epithelium in hyperplastic growth, with no evidence of focal or malignant tumours. These observations show that in addition to neu, myc, ras and ret transgenes, mos, a member of the protein‐serine/threonine kinase family of oncogenes, can induce Harderian gland hyperplasia, thus revealing an unusual response by this organ to various classes of oncogenes. Analysis of fos, jun, myc and ets oncogene RNA in mos‐induced hyperplastic Harderian glands shows that there are no consistent changes in the level of expression of these oncogenes, suggesting that mos acts via a mechanism other than by increasing the expression of these genes.
Enzymatic basis of sugar structures of α‐fetoprotein in hepatoma and hepatoblastoma cell lines: Correlation with activities of α1–6 fucosyltransferase and N‐acetylglucosaminyltransferases III and V
Authors: Ohno Masao , Nishikawa Atsushi , Koketsu Masatoshi , Taga Hiroko , Endo Yasuo , Hada Toshikazu , Higashino Kazuya , Taniguchi Naoyuki
Published: February 2007

α‐Fetoproteins (AFPs) were purified from 2 hepatoma cell lines (Hep G2 and HuH‐7) and a hepatoblastoma cell line (HuH‐6), and the structures of pyridylaminated (PA) derivatives of their sugar chains were analyzed by HPLC. Simultaneously, the activities of αl‐6 fucosyltransferase (αl‐6FT) and N‐acetylglucosaminyltransferase III (GnT‐III), IV(GnT‐IV) and V(GnT‐V) were assayed in these cell lines. For all 3 cell lines the major sugar chain detected was a fucosylated biantennary structure. Hep G2 cells contained a high level of GnT‐V, which catalyzes the formation of a tri'‐antennary structure, and in fact a substantial percentage of the AFP sugar chains in these cells had the tri'‐antennary structure. αl‐6FT was also high, and fucosylated tri' structures were detected, which suggests that high activities of transferases affect the AFP sugar chains. In HuH‐6 cells, GnT‐III, which catalyzes the formation of bisecting GIcNAc, was elevated. Correspondingly, a fucosylated, bisected biantennary structure was found as a major sugar chain. In the HuH‐7 cell line, the contents of bisecting GIcNAc and tri' structure were low and neither GnT‐III nor GnT‐V was elevated. These data indicate that the sugar structures of AFP in these cell lines correlate well with the activities of αl‐6 FT, GnT‐III and GnT‐V.
Stimulation of human prostatic carcinoma tumor growth in athymic mice and control of migration in culture by extracellular matrix
Authors: Passaniti Antonino , Isaacs John T. , Haney Joseph A. , Adler Scott W. , Cujdik Timothy J. , Long Peter V. , Kleinman Hynda K.
Published: February 2007

The tumorigenicity, migration, growth and invasiveness of certain tumor cells is stimulated by basement membranes. Here we have examined the effect of Matrigel, an extract of basement membrane proteins, on the behavior of several prostate cancer cell lines, testing their growth and invasiveness in vitro and in vivo. Cells of the Tsu‐prI line were more invasive than PC‐3, Du‐145, or LNCaP cells. Peptide inhibitors implicated laminin in the migration and invasion of these cells. When these cells were suspended in Matrigel and injected into nude mice, their growth was greatly enhanced, since large tumors formed in athymic nude mice whereas virtually no tumors were observed in the absence of Matrigel. The growth of a slowly growing line, LNCaP, was increased by exogenous basic fibroblast growth factor when injected with Matrigel. A laminin cell adhesion peptide, YIGSR, was a potent inhibitor of Matrigelstimulated tumor growth implicating cell‐laminin interactions in this process. These results suggest that tumor growth of prostate adenocarcinoma cells may be dependent both on cellular growth factors and on cell‐matrix interactions mediated by laminin which facilitate the development of transplanted tumors in nude mice.
Fibrosis in immune control of mammary‐tumor growth
Authors: Vaage Jan
Published: February 2007

In malignancies, an interruption of growth (dormancy) is sometimes observed. In the immunogenic mammary carcinoma MC2, dormancy followed by regression after a period of growth was observed in 18% of s.c. implants in normal mice. Dormant implants removed for histologic examination were invariably found to be completely surrounded by a highly fibrous stroma. Fibrosis was enhanced in immunized mice, and reduced in immuno‐deficient mice. Surgical disruption of the fibrous capsule around dormant tumors early (19 ± 3 days) in the immune response led more frequently to resumed growth, while later (32 ± 3 days) disruption of the capsule led more frequently to complete regressions. This showed that fibrous capsules that could destroy tumors could also shield them against well‐developed systemic immune mechanisms.
Levels of expression of breast epithelial mucin detected by monoclonal antibody BrE‐3 in breast‐cancer prognosis
Authors: Ceriani Roberto L. , Chan Curtis M. , Baratta Frank S. , Ozzello Luciano , Derosa Carolyn M. , Habif David V.
Published: February 2007

Taking into consideration the relationship of breast neoplasia with recent knowledge obtained on the molecular structure and biosynthesis of the breast epithelial mucin, an epitope on this molecule detected by monoclonal antibody (MAb) BrE‐3 was chosen as a marker to study the correlation of expression of the mucin and prognosis in infiltrating ductal carcinomas of the breast. Strong statistical validation was obtained in the use of BrE‐3 in immunohistochemical procedures where scores for the expression of the mucin on paraffin‐embedded sections of the primary breast tumors were studied. Four different immunohistochemical variables measuring levels of expression (intensity or prevalence) in cytoplasm or membrane were obtained for each of 227 patients' breast tumors and subjected to KaplanMeier univariate and Cox proportional‐hazards multi‐variate analysis. Additionally, traditional prognostic variables for breast‐cancer prognosis (grade of differentiation, age, tumor size, axillary‐lymph‐node involvement and estrogen receptors) were subjected to identical analyses. In uni‐variate analysis, low cytoplasmic intensity, high membrane prevalence, and high membrane intensity of mucin expression were each found to be significantly associated with good prognosis in relation to both survival or relapse time. In multi‐variate analysis, all 4 immunohistochemical parameters were significantly associated with both survival and relapse time in these patients. Among the traditional variables, 3 (axillary‐node involvement, grade of differentiation and tumor size) were also found to be statistically significant at the uni‐variate and multi‐variate level. A multi‐variate analysis of the combined immunohistochemical and traditional variables identified the 4 immunohistochemical parameters, tumor size and axillary‐node involvement as having the highest level of association with either survival or relapse time. These variables were then combined and served to define a prognostic model [ILCPS(Comb)], which was found to have the capacity to separate the patient population studied into 4 prognostic groups in terms of survival and 3 groups in terms of relapse. As expected, the ILCPS(Comb) was shown to have a higher level of prognostic association with both survival and relapse than the individual variables themselves, the traditional variables together or the immunohistochemical variables together. Our approach develops a theoretical framework and a statistical model, employing levels of expression of the breast epithelial mucin and 3 traditional variables, which identifies, in terms of prognosis, distinct sub‐populations of patients with infiltrating breast carcinoma with defined risk functions.
A collaborative case‐control study of nutrient intake and pancreatic cancer within the search programme
Authors: Howe G. R. , Ghadirian P. , de Mesquita H. B. Bueno , Zatonski W. A. , Baghurst P. A. , Miller A. B. , Simard A. , Baillargeon J. , de Waard F. , Przewozniak K. , McMichael A. J. , Jain M. , Hsieh C. C. , Maisonneuve P. , Boyle P. , Walker A. M.
Published: February 2007

Case‐control studies of pancreatic cancer were conducted in 5 populations with moderate to high rates and differing dietary practices, using a common protocol and questionnaire. Comprehensive diet histories were completed for a total of 802 cases and 1669 controls identified in Adelaide (Australia), Montreal and Toronto (Canada), Utrecht (The Netherlands) and Opole (Poland). Positive associations were observed with intake of carbohydrates and cholesterol, and inverse associations with dietary fiber and vitamin C. These relationships were generally consistent among the 5 studies, and showed statistically significant and generally monotonic dose‐response relationships. The relative risks for highest vs. lowest quintile of intake were estimated for carbohydrates to be 2.57 (95% confidence interval 1.64–4.03), cholesterol 2.68 (1.72–4.17), dietary fiber 0.45 (0.30–0.63), and vitamin C 0.53 (0.38–0.76). The consistency, strength, and specificity of these associations provides evidence for the hypothesis that some or all of these dietary factors may alter the risk of pancreatic cancer.
Induction of growth suppression and modification of gene expression in multi‐drug‐resistant human glioblastoma multiforme cells by recombinant human fibroblast and immune interferon
Authors: Moulton Thomas A. , Jiang Hongping , Guarini Ludovico , Fetell Michael R. , Fisher Paul B.
Published: February 2007

The combination of recombinant human fibroblast (IFN‐β) and immune (IFN‐γ) interferon induces enhanced growth suppression and modifies the antigenic phenotype in parental and multi‐drug‐resistant (MDR) human glioblastoma multiforme (GBM) cells. The present study was conducted to explore the mechanism underlying this cooperative interaction between interferons in inducing growth suppression in MDR‐GBM cells. For this analysis we have utilized 2 MDR‐GBM cell lines which display a differential sensitivity to growth suppression when exposed to IFN‐β or IFN‐γ. GBM‐18‐B3 (MDR) cells are more sensitive to growth inhibition by IFN‐γ than by IFN‐β, whereas GBM‐18‐A3 (MDR) cells are inhibited to a greater degree by IFN‐β than by IFN‐γ. In both cell types, however, growth is suppressed to a greater degree by the combination of interferons than by equivalent concentrations of either type of interferon used alone. Growth suppression induced by the interferons, alone or in combination, was not associated with comparable changes in the steady‐state level of MDRI mRNA. In addition, the anti‐proliferative effect of interferon was similar in GBM‐18 (MDR) cells grown in the presence or absence of colchicine. GBM‐18‐A3 and GBM‐18‐B3 cells differed in their de novo and interferon‐inducible expression levels of IFN‐β‐responsive genes, isg‐15 and isg‐54. In contrast, both cell types responded in a similar manner with respect to expression of the IFN‐γ‐responsive gene, HLA Class II (HLA‐DRβ), and HLA Class I, fibronectin and ICAM‐1, No further increase in expression of any of the genes was observed which was unique to the combination of interferons.
Loss of HLA class‐I alleles, heavy chains and β2‐microglobulin in colorectal cancer
Authors: Kaklamanis Loukas , Gatter Kevin C. , Hill Ann B. , Mortensen Neil , Harris Adrian L. , Krausa Peter , McMichael Andrew , Bodmer Julia G. , Bodmer Walter F.
Published: February 2007

Using immunohistochemical methods, we have analysed colorectal biopsies of normal mucosa, metaplastic polyps (5 cases), adenomas (15 cases) and adenocarcinomas (70 cases) with 13 monoclonal antibodies (MAbs) to allelic products of the HLA‐A, B, C loci. Nine of the 70 carcinomas showed total loss of HLA Class‐I molecules due to an underlying defect regarding not only the expression of β2‐microglobulin (·β2‐m), but also the heavy chains of HLA A, B and C loci, or both. Much commoner was a loss of one or more Class‐I alleles as follows: Al/Aw36 (completely lost in 4 of 29 cases and focally lost in another 2), A2 (in 1 of 37 cases), A3 (in 2 of 14 cases), A11/28/31/33 (in 3 of 11 cases), B7 (in 3 of 13 and focally in 1 other case), B17 (in 1 case), Bw4 (in 8 of 45 and focally in another 6), Bw6 (in 9 of 62 and focally in another 3). Focal selective loss (Bw6 and a combined A1‐Bw6), was observed in 2 adenomas. Normal colonic mucosa, as well as stromal and lymphoid cells present between the neoplastic glands, were studied in each case as a control. A particular allele was only considered to be lost by the malignant cells if it was still expressed on these adjacent tissues.
Risk of lung cancer among cigarette and pipe smokers in Southern China
Authors: Lubin Jay H. , Jun‐Yao Li , Xiang‐Ghen Xuan , Kan Cai Shu , Qing‐Sheng Luo , Shen Yang Li , Jian‐zhang Wang , Lan Yang , Blot William J.
Published: February 2007

Studies in Shanghai and in north‐east China indicate that cigarette smoking is a major contributor to the high rates of lung cancer in those areas, but doubts persist regarding the influence of cigarette use on lung cancer rates in other areas of China. In addition, the risk of lung cancer associated with other methods of tobacco consumption—in particular, the use of bamboo water‐pipes and long‐stem pipes—is uncertain. A population‐based case‐control study of 427 male lung cancer patients residing in a mining area of Southern China and 1,011 controls was carried out to address this and other issues. Of these patients, 63% smoked cigarettes and (water and longstem) pipes; 17% and 14% smoked only cigarettes or pipes, respectively; and 6% did not smoke. Compared to non‐smokers, smokers of cigarettes only, smokers of pipes only and mixed smokers were at increased risk; OR = 2.6 (95% CI 1.1–6.2), 1.8 (95% CI 0.8–4.2) and 4.1 (95% CI 2.3–9.2), respectively. Risk increased with duration of tobacco use; however, the rate of increase with years of cigarette use was significantly greater than for years of pipe use (p = 0.03). In addition, risks increased 8‐fold in the highest quartile of number of cigarettes per day compared to non‐cigarette smokers vs. 2.3‐fold for the highest quartile of number of liang (50 g) smoked per month compared to non‐pipe‐smokers; the trends in the ORs differed significantly (p < 0.001). Results suggest that, in this area of China, tobacco use is an important cause of lung cancer, and that smoking cigarettes may be more deleterious than smoking pipes (primarily water pipes).
Increased expression of αIIb β3 integrin in subpopulations of murine melanoma cells with high lung‐colonizing ability
Authors: Chang Y. S. , Chen Y. Q. , Timar J. , Nelson K. K. , Grossi I. M. , Fitzgerald L. A. , Diglio C. A. , Honn K. V.
Published: February 2007

Four subpopulations of B16 amelanotic melanoma cells, possessing different abilities to induce platelet aggregation (TCIPA) and to form lung colonies, were isolated by centrifugal elutriation. The expression of αIIb, α3 and α5β1 integrins was examined in the 4 subpopulations in order to determine the relationship between integrin receptor expression and tumor‐cell metastatic potential. The mRNA of αIIb, α5, β1 and β3 was detectable in the 4 subpopulations by Northern blotting. A gradual increase in mRNAs and cell‐surface immunoreactivity of the αIIbβ3 receptor, but not in their gene copies, was observed from the low to the high metastatic subpopulations. The ability of tumor cells to adhere to fibronectin and subendothelial matrix (SEM) increased in parallel. In the high metastatic cells, the αIIbβ3 receptors, but not the α5β1 receptors, were localized to focal adhesion plaques. Incubation of the high metastatic cells with αIIbβ3‐specific antibodies reduced their matrix adhesion, TCIPA and lung‐colonizing abilities. In contrast, in the low met‐ astatic cells, SEM adhesion and lung‐colony formation were not affected by anti‐αIIbβ3 antibody treatment. Incubation of either the low or the high metastatic subpopulation with an α5β1‐specific antibody had no effect in vitro and showed a slight inhibition of lung colonization in vivo. Our results suggest that several phenotypic characteristics of the enhanced metastatic potential of B16a subpopulations may be mediated by increased expression of αIIbβ3 receptors and that expression of these receptors may be regulated at the transcriptional level.
Comparative anti‐viral and anti‐proliferative activity of PGA1 and PGJ2 against HTLV‐I‐infected MT‐2 cells
Authors: D'Onofrio C. , Amici C. , Puglianiello A. , Faraoni I. , Lanzilli G. , Santoro M. G. , Bonmassar E.
Published: February 2007

Prostaglandin (PG) A and J exert anti‐viral and anti‐proliferative effects in a number of experimental models. In particular, multiple treatments with PGAs prevent in vitro the clonal selection of HTLV‐l‐infected and potentially transformed cord‐blood‐derived mononuclear cells. Proliferation of HTLV‐I‐infected leukemic T cells is refractory in most cases to conventional anti‐blastic therapy. We examined whether these cyclo‐pentenone PGs might control cell proliferation and/or alter virus replication also in HTLV‐l‐transformed cells. We show that PGA1 and PGJ2 can exert powerful control of proliferation of the HTLV‐l‐immortalized, virus‐producing MT‐2 cell line, in a concentration‐dependent fashion. Cells were preferentially arrested at the G1/S interface by treatment with PGA1 or PGJ2 without any detectable cellular toxicity. The anti‐proliferative effect of PG treatment was independent of the growth phase of MT‐2 cells, since both asynchronous and synchronous cells were sensitive to treatment. This effect was accompanied by an increase in the synthesis of a 70 kDa heat‐shock protein (HSP70). However, synthesis of HSP70 was induced to a much greater extent by PGJ2 than by PGA1 at the same concentration. Neither PGA1 or PGJ2 inhibited the transcription of HTLV‐I in MT‐2 cells, but treatment with PGJ2, and not with PGA1, moderately inhibited the synthesis of viral proteins, i.e., p40 Tax and p19 core proteins. Moreover, infection of recipient K562 cells was significantly inhibited after pre‐treatment of MT‐2 cells with PGJ2 14 hr before or co‐treatment at the onset of the co‐culture with K562 cells. This effect was not obtained when MT‐2 cells were repeatedly pre‐treated with PGJ2 for I week before co‐culturing. This suggests that reduced infection could be related to impairment of some step in virus‐transmission phase.
High in vitro‐in vivo correlation of drug response using sponge‐gel‐supported three‐dimensional histoculture and the MTT end point
Authors: Furukawa Toshiharu , Kubota Tetsuro , Watanabe Masahiko , Takahara Tetsuya , Yamaguchi Hiroshi , Takeuchi Tooru , Kase Suguru , Kodaira Susumu , Ishibiki Kyuya , Kitajima Masaki , Hoffman Robert M.
Published: February 2007

The in vitro sponge‐gel‐supported three‐dimensional histoculture chemosensitivity assay (Hoffman assay) allows the in vivo‐like culture of human tumors. In this study, the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H tetrazolium bromide (MTT) end point was applied to the Hoffman assay in an attempt to increase in vitro‐in vivo correlation. The chemosensitivities of 16 human tumor lines were determined in vitro by the histoculture assay, and retrospectively correlated to their in vivo chemosensitivity as xenografts in nude mice. The in vitro test was considered to be positive if tumor‐cell MTT reduction activity was lowered by more than 50%. The cutoff drug concentrations to determine sensitivity in vitro were determined for mitomycin C, doxorubicin, 5‐fluorouracil and cisplatin. Using these cutoff drug concentrations in vitro we found, as a function of time of exposure, a strong correlation between serum drug concentrations found in nude mice given maximum tolerated doses and drug concentrations found in the histoculture media in vitro, thereby establishing a relationship between the amounts of drugs to which tumors were exposed in vivo and in vitro. The overall correlation rate of the efficacy results of the drug‐response assay to in vivo chemosensitivities was 89.8%, with 90.0% true‐positive and 89.7% true‐negative rates, 81.7% sensitivity and 94.6% specificity, thereby indicating potential clinical use for tumor histoculture with the MTT end point.