bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of PAP and PSA gene expression in prostatic hyperplasia and prostatic carcinoma using northern‐blot analyses, in situ hybridization and immunohistochemical stainings with monoclonal and bispecific antibodies
Authors: Hakalahti Leena , Vihko Pirkko , Henttu Pirkko , Vihko Reijo , Autio‐Harmainen Helena , Soini Ylermi
Published: February 2007

In this report we have investigated levels of prostatic acid phosphatase (PAP) and prostate‐specific antigen (PSA) gene expression in prostatic carcinoma (Ca) and benign prostatic hyperplasia (BPH) specimens. Northern‐blot analyses of total prostatic mRNA indicated that there was a tendency towards lower amounts of PAP mRNA and PSA mRNA in the Ca specimens than in the BPH specimens, although, because of the great variation in the expression levels of both mRNAs, these differences were not statistically significant. In situ hybridization analyses clearly showed that both PAP and PSA mRNAs were confined to the columnar epithelial cells and that stromal cells were devoid of these mRNAs. In addition, PAP and PSA mRNAs were more abundant in BPH tissue than in adjacent Ca tissue within the same specimen. The levels of PAP and PSA enzymes were analyzed immunohistochemically using a bispecific antibody having high affinity for both PAP and PSA, and the results were compared with those obtained using monoclonal anti‐PAP and anti‐PSA antibodies. All 3 antibodies stained only epithelial cells and BPH tissue consistently gave more intense staining than Ca tissue. Furthermore, the anti‐PSA and the bispecific anti‐PAP‐PSA antibodies stained well or moderately differentiated Ca tissues more strongly than poorly differentiated Ca tissues. No PSA staining was detected in 3 and no PAP staining in 5 of the moderately or poorly differentiated carcinomas (grades II or III). Our results show that, in comparison with BPH tissue, prostatic Ca tissue is associated with significantly lower levels of mRNAs coding for the prostatic marker enzymes PAP and PSA, as well as with lower concentrations of these enzymes. Furthermore, dedifferentiation of prostate Ca is associated with a decrease in the level of intraprostatic PSA.
PCR detection and typing of genital papillomavirus in a new brunswick population
Authors: Kurz J. , Mitra K. , Adam R. , Miao X. , Coombs D. H. , Krause M. O. , Mackay J. S. , Isa N. N.
Published: February 2007

We have used a broad range of primers for HPV detection, using the polymerase chain reaction (PCR) so as to compare PCR typing of HPV with the results of cytological diagnosis in a New Brunswick population referred to the out‐patient clinic of the Saint John Regional Hospital. The primers selected were found to be capable of amplification with high efficiency, therefore we did not perform further hybridization analysis for specific identification of HPV types. Amplification of selected fragments for detection of HPV 6, 11, 16, 18, 31 and 33 was obtained from cervical swabs collected from 154 patients. Microscopic examination was performed in duplicate samples and the results compared with the DNA‐typing analysis. HPV of any of the above types was detected in 43 out of 154 patients. Among these, 32 patients showed single or multiple infections with “high‐risk” HPV strains 16, 18, 31 or 33. Cytologically normal or atypical samples with any of the HPV types tested amounted to 17%, but increased to 56% in patients with CIN I, and to 100% in patients with CIN II or III. Prevalence of “high‐risk” types alone increased from 15% and 10%, for normal and atypical cases respectively, to 48% for CIN I, 75% for CIN II and 100% for CIN III. Our results indicate that HPV detection and typing by this simple procedure can be a valuable indicator of cancer progression and thus can help to identify individuals at high risk in pre‐malignant stages of the disease.
CIS‐diamminedichloroplatinum(II) inhibits p34cdc2 protein kinase in human lung‐cancer cells
Authors: Nishio Kazuto , Fujiwara Yasuhiro , Miyahara Yuki , Takeda Yuichiro , Ohira Tatsuo , Kubota Naohiro , Ohta Sei , Funayama Yasunori , Ogasawara Hayato , Saijo Nagahiro , Ohata Masahiro
Published: February 2007

cis‐Diamminedichloroplatinum(II) (CDDP) induced G2‐phase arrest in PC‐9 human cancer cells. To elucidate how CDDP acts on cell‐cycle regulation, we analyzed the effect of CDDP on cell‐cycle regulators such as p34cdc2 protein kinase. p34cdc2 protein kinase activity was maximum in G2 phase and decreased after G2/M transition in synchronized PC‐9 human lung cancer cells. Evidence for a phosphorylated p34cdc2 protein kinase complexed with cyclin B was obtained from cells in G2 phase and the p34cdc2 protein kinase appeared to be dephosphorylated at M phase. After exposure to CDDP in G, phase, PC‐9 cells were arrested in G2 phase. The activation of p34cdc2 protein kinase was inhibited by CDDP. Cyclin A and wee‐1 kinase were not affected by the exposure to CDDP. Cyclin B was degraded in M phase in PC‐9 cells. Exposure to CDDP did not affect the degradation of cyclin B. Our data suggest that the effect of CDDP on cell‐cycle phase might be regulated by the dephosphorylation of p34cdc2 protein kinase. To determine whether the p34cdc2 protein kinase is a primary target for CDDP, we examined the direct effect of CDDP on tyrosine dephosphorylation of p34cdc2 protein kinase in cellular extracts. Cell lysates from synchronized PC‐9 in G2 phase were immunoprecipitated with p 13‐Sepharose beads. In vitro dephosphorylation of phosphotyrosine of p34cdc2 protein kinase was observed after exposure to okadaic acid in a concentration‐dependent manner. The dephosphorylation of p34cdc2 protein kinase by okadaic acid was inhibited by CDDP. We hypothesize that inhibition of p34cdc2 dephorphorylation by CDDP is important for its growth‐inhibiting properties.
The influence of angiogenesis inhibitor AGM‐1470 on immune system status and tumor growth in vitro
Authors: Schoof Deric D. , Obando J. Antonio , Cusack James C. , Goedegebuure Peter S. , Eberlein Timothy J. , Brem Harold
Published: February 2007

The synthetic angiogenesis modulator‐1470 O‐(chloroacetyl‐carbomoyl, AGM‐1470) is a potent inhibitor of neovascularization. We have investigated the potential influence of this inhibitor in tumor immunobiology using both in vivo and in vitro models. Mice given a single tail‐vein injection of tumor cells were later treated with AGM‐1470 by s.c. injection. After tumor injection, the lungs were evaluated for macroscopic tumor nodules. AGM‐1470 significantly reduced the development of macroscopic pulmonary disease but did not eliminate disease. However, tumor‐bearing mice treated with AGM‐1470 had significantly reduced spleen weight compared to controls. To determine if the observed decrease in spleen weight in the treated animals was associated with immunosuppression, we studied the possible immunomodulatory effects of AGM‐1470. AGM‐1470 induced no changes in spleen‐cell viability compared to controls. However, addition of angioinhibin at the beginning of IL‐2‐induced spleen‐cell activation significantly inhibited the development of NK‐mediated tumor‐cell killing. Similarly, splenic T‐cell proliferation induced by a mitogenic monoclonal antibody to murine T cells was significantly inhibited when activated in the presence of AGM‐1470. The in vitro studies were extended by evaluation of immune system status in tumor‐bearing mice treated with AGM‐1470. In vivo therapy with AGM‐1470 did not significantly change the mean splenic lymphocyte counts and CD4/CD8 ratios from control values. In addition, the induction of splenic NK‐mediated tumor killing with IL‐2 as well as mitogen‐induced T‐cell activation was not significantly different from control values. These results suggest that AGM‐1470 inhibits tumor growth by blocking neovascularization and may, under certain conditions of drug administration, inhibit immune system function.
Flunarizine as a modulator of doxorubicin resistance in human colon‐adenocarcinoma cells
Authors: Silvestrini Rosella , Zaffaroni Nadia , Costa Aurora , Orlandi Linda , Villa Raffaella , Hendriks Hans R.
Published: February 2007

The potential of the calcium‐entry blocker flunarizine in modulating the cytotoxicity of doxorubicin was investigated in human colon‐adenocarcinoma cell lines sensitive to (LoVo) or with experimentally induced resistance (LoVo/DX) to doxorubicin. Exposure to 1 to 2 m̈g/ml flunarizine for intervals of up to 24 hr did not affect cell survival in either line. Simultaneous exposure to flunarizine and doxorubicin for 1 hr selectively enhanced doxorubicin activity in the resistant cell line and not in the sensitive cell line. In particular, the doxorubicin concentration able to reduce cell survival by 50% dropped to one third. Moreover, simultaneous exposure to flunarizine significantly increased intracellular doxorubicin accumulation, as evaluated by fluorescence spectrophotometry. Again, flow‐cytometric analysis showed hyperpolarization of the membrane in resistant cells, starting from 15 min of exposure to 2 m̈g/ml flunarizine. Finally, in LoVo/DX cells, which normally express gpl70, a 24‐hr treatment with flunarizine markedly reduced the immuno‐reactivity of cells with 2 monoclonal antibodies (MAb57 and MRK16) directed against different external epitopes of the glycoprotein. The results from our study indicate the ability of flunarizine to positively modulate doxorubicin‐resistance in human colon‐adenocarcinoma cells expressing the multidrug‐resistance phenotype.
Importance of mevalonate‐derived products in the control of HMG‐CoA reductase activity and growth of human lung adenocarcinoma cell line a549
Authors: Bennis Faïza , Favre Gilles , Gaillard Francis Le , Soula Georges
Published: February 2007

HMG‐CoA reductase catalyzes the synthesis of mevalonate, a crucial intermediate in the biosynthesis of cholesterol and non‐sterol isoprenoid compounds essential for cell growth. The HMG‐CoA reductase activity of the A549 tumor cell line is higher than that of normal human fibroblasts. This deregulation in mevalonate needs was not due to an alteration in the activated state of the enzyme by short‐term regulation. We show that the HMG‐CoA reductase in A549 cell line was subject to a multivalent feedback control. A high fraction (40%) of the reductase activity was devoted to non‐sterol products. In contrast, normal fibroblasts had only 15–20% of the reductase activity that generated non‐sterol products. We also show that cholesterol and at least one of the non‐sterol products are necessary for optimal cell growth of A549 cells. Our data strongly suggest that A549 cells produce more non‐sterol substances which may be related to increased requirements of mevalonate for upregulated cell growth.
Role of cell cholesterol in modulating vincristine uptake and resistance
Authors: Pallarés‐Trujillo J. , Domènech C. , Grau‐Oliete M. R. , Rivera‐Fillat M. P.
Published: February 2007

The relationship between cell‐membrane permeability to vincristine and cholesterol/phospholipid levels was studied in L5I7SY murine leukemic lymphoblasts and in 2 multidrug‐resistant cell sublines, VCR/P60 and VCR/P200, which expressed increasing levels of vincristine resistance. The uptake of 3H‐vincristine was measured in all cell lines and in cholesterol‐depleted and ‐reloaded L5178Y and VCR/P200 cells. The initial rate of drug entry in resistant cells was lower than that measured in the parental cell line and it decreased as the relative resistance increased. An increment of cholesterol content, characterized in resistant cells, was directly proportional to the relative resistance to vincristine. Cholesterol depletion in both sensitive and resistant cells resulted in an increase in the rate of vincristine uptake, which reverted to the respective basal levels when each cell line was cholesterol‐reloaded. The rate of drug uptake was inversely correlated with the molar ratio of cholesterol to phospholipids. Although both VCR/P cell sublines, but not the sensitive parental cells, expressed the P‐glycoprotein in their plasma membrane, there were no differences in drug efflux and retention between resistant and parental cells. These results indicate that cholesterol modulates the permeation of vincristine through the plasma membrane and strongly suggest that increased levels of cholesterol/ phospholipid account for the lower drug accumulation and greater resistance in these multidrug‐resistant cells.
Specific expression of the pancreatic‐secretory‐trypsin‐inhibitor (PSTI) gene in hepatocellular carcinoma
Authors: Ohmachi Yoshitaka , Murata Atsuo , Yasuda Tadashi , Yasuda Takushi , Monden Morito , Mori Takesada , Ogawa Michio , Matsubara Kenichi
Published: February 2007

Twenty hepatocellular carcinomas (HCC) were analyzed by Northern blotting to test the expression of pancreatic secretory trypsin inhibitor (PSTI). This gene was expressed in all HCCs, but not in other tumors, including mammary, thyroid, pulmonary and ovarian cancers. Some gastric and colonic cancers weakly expressed PSTI. Among cell lines examined in a similar manner, PSTI was expressed in all of 4 derived from hepatoma. On the other hand, among 15 cell lines derived from cancers other than hepatoma, only 3, derived from pancreatic, colonic and gastric cancers, weakly expressed PSTI. A CAT assay using a deletion set of the 5′ region from the cloned PSTI gene has shown that in hepatoma cell lines, the expression of this gene is dependent on the presence of 2 regulatory regions that include an IL‐6 responsive element and an AP‐1‐binding site. However, in non‐hepatoma cell lines, the 2 regulatory regions are not necessary for expression. The blood level of PSTI in 27 patients with HCC was significantly increased, and it was positively correlated with tumor size, suggesting that specific expression of PSTI in HCC causes this effect and that elevated blood level of PSTI without inflammation indicates the presence of HCC.
Intratumoral microvessel density and L53 protein: Correlation with metastasis in head‐and‐neck squamous‐cell carcinoma
Authors: Gasparini Giampietro , Weidner Noel , Maluta Sergio , Pozza Franco , Boracchi Patrizia , Mezzetti Maura , Testolin Alessandro , Bevilacqua Pierantonio
Published: February 2007

Squamous‐cell carcinoma of the head and neck includes a heterogeneous group of tumours of the upper air and food passages for which prognosis is difficult to assess. In fact, patients in comparable stages may have diverse clinical courses and responses to similar treatments. In order to better define the prognosis of each patient there is therefore a need to identify novel biological markers which reflect more accurately growth rate, progression and metastatic potential of each tumour. We assessed whether metastases correlate with micro‐vessel counts (i.e. intratumoral vascularity) using the CD‐31 monoclonal antibody (MAb) and p53 mutant protein expression, determined in the primary by immunocytochemical methods in 70 patients with locally advanced head and neck cancer. Patients were treated with concurrent chemo‐radiotherapy; 50 of these presented loco‐regional node metastasis at diagnosis whereas 3 cases, initially node‐negative, developed distant metastasis during the period of observation. No feature was predictive for objective response to treatment. The overall mean and median blood vessel density at “hot spots” was 37.42 and 36, respectively, and 57% of the tumours expressed p53 mutant proteins. These 2 biological markers were significantly associated. Patients with metastases (loco‐regional and distant) had a significantly higher mean blood‐vessel density than those without tumour spread. Also, patients with p53‐positive (+/ + +) tumours had a significantly higher incidence of metastasis than those with negative ones. Multivariate analysis showed that both vascularity and stage, but not p53 expression, are significant and independent predictors of metastasis in this series.
Expression of αB‐crystallin in human brain tumors
Authors: Aoyama Akira , Steiger Rudolf H. , Fröhli Erika , Schäper Reinhold , Deimling Andreas , Wiestler Otmar D. , Klemenz Roman
Published: February 2007

We have previously shown that αB‐crystallin is a heat‐shock protein which specifically accumulates in response to the expression of c‐Ha‐ras and v‐mos oncogenes in mouse NIH 3T3 fibroblasts. Elevated levels of αB‐crystallin mRNA or protein were shown to be associated with pathological conditions of the brain. Therefore, we have examined the expression of αB‐crystallin in normal human brains and brain tumors by Western blot analysis. αB‐crystallin is moderately expressed in adult but not fetal brain. Elevated levels of αB‐crystallin expression are observed in glial tumors such as astrocytoma, glioblastoma multiforme, and oligodendroglioma. αB‐crystallin in these tumors is predominately unphosphorylated. High amounts of accumulated aB‐crystallin in astrocytic tumors are preferentially found in the more aggressive stages. Glioblastoma multiforme is exceptional in that high αB‐crystallin expression is observed in only one half of the analyzed samples whereas no aB‐crystallin could be detected in the other. These results indicate that αB‐crystallin may be a useful biochemical marker for studying the pathogenesis of various human brain tumors.
Helicobacter pylori infection in japanese patients with adenocarcinoma of the stomach
Authors: Blaser Martin J. , Kobayashi Kensuke , Cover Timothy L. , Cao Ping , Feurer Irene D. , Pérez‐pérez Guillermo I.
Published: February 2007

To examine the association of Helicobacter pylori infection with adenocarcinoma of the stomach in Japanese patients, we studied 29 patients and 58 matched controls. Ascertainment of H. pylori status was based on the presence of specific IgG to H. pylori. For the entire group, an association of this infection with gastric adenocarcinoma was suggested but not statistically significant. For patients in a putatively high‐risk subgroup (non‐cardia tumors and age ≤70 years), the association was significant. Assays detecting serum IgA to whole H. pylori cells and cytotoxin, IgG to cytotoxin and Hp54K (the heat‐shock protein homolog) and serum neutralization of cytotoxin activity each showed clear differences between H. pylori‐infected and uninfected persons in this population. However, for none of these assays was there a significant difference between values for H. pylori‐infected persons with or without gastric cancer. Thus, while H. pylori infection was associated with non‐cardia gastric cancer in Japanese persons ≤70 years of age, use of these additional serologic markers did not define additional factors that might be associated with increased risk.
Gamma‐globulin modulates growth of EBV‐derived B‐cell tumors in scid mice reconstituted with human lymphocytes
Authors: Abedi Mohammad R. , Christensson Birger , Al‐Masud Salahuddin , Hammarström Lennart , Smith C. I.
Published: February 2007

The effect of weekly γ‐globulin injection on the development of human B‐cell tumors was studied in 120 mice with severe combined immunodeficiency (SCID). The mice were injected intraperitoneally (i.p.) with human peripheral mononuclear cells (PBMC) from 6 different Epstein‐Barr virus (EBV)‐seropositive donors. Animals repopulated with cells from 5 donors received γ‐globulin or saline for 20 weeks and were followed up to 24 weeks after reconstitution. A delay in the appearance of fatal EBV‐derived human B‐cell tumors was noticed in the γ‐globulin‐treated groups as compared to the controls. In a separate experiment, the effect of γ‐globulin treatment during the initial 4 weeks after reconstitution was compared to treatment from week 5 to week 8 as well as to a continuous 20‐week treatment. The results from this experiment showed that B‐cell tumor growth could be prevented just as efficiently when the animals were treated only during the first 4 weeks. In contrast, no preventive effect was seen when the first γ‐globulin dose was given at the beginning of week 5 after reconstitution. Our results indicate that γ‐globulin reduces the frequency of EBV‐derived B‐cell tumor development and suggest that SCID mice repopulated with human cells represent a useful in vivo model for evaluation of the prophylactic and/or therapeutic effects of immunomodulatory treatments in lymphoproliferative disorders associated with immunosuppression.
Effect of buthionine sulfoximine on PtII and PtIV drug accumulation and the formation of glutathione conjugates in human ovarian‐carcinoma cell lines
Authors: Mistry Prakash , Loh Swee Y. , Kelland Lloyd R. , Harrap Ken R.
Published: February 2007

Glutathione (GSH) has often been implicated in the mechanism of resistance to platinum anti‐cancer drugs. It has been suggested that GSH may reduce the cytotoxicity of these drugs by forming inactive conjugates and by enhancing the repair of DNA‐platinum crosslinks. In the present study we have examined the effect of D, L‐buthionine‐S, R‐sulfoximine (BSO) pretreatment on the accumulation of platinum in a sensitive (CH I) and 2 relatively resistant (SKOV‐3, HX/62) human ovarian‐carcinoma cell lines following exposure to PtII‐ (cisplatin, carboplatin) and PtIV‐drugs (tetraplatin). The metabolism of cisplatin and tetraplatin (particularly the extent of platinum‐GSH conjugate formation) in the presence and absence of BSO pre‐treatment was also examined in these cell lines. BSO pretreatment reduced the accumulation of PtII but not that of PtIV drugs in the relatively resistant SKOV‐3 and HX/62 cell lines. It had no effect on the accumulation of either class of drugs in the sensitive CHI cells. Metabolism studies with cisplatin showed that the SKOV‐3 and HX/62 cells, which contained 2‐ to 3‐fold higher levels of GSH, were able to inactivate a greater proportion of cellular cisplatin, by the formation of platinum‐GSH conjugates, than the CH I cells. A significant inhibition in formation of these conjugates, by BSO‐induced depletion of cellular GSH (over 80%), did not, however, increase cisplatin concentration in the resistant cells. In contrast, a small increase in cisplatin concentration was observed in the sensitive cells following BSO pre‐treatment. Comparison of cisplatin and tetraplatin metabolism in the SKOV‐3 cells indicated that a greater proportion of the latter drug was inactivated by formation of GSH conjugates. BSO‐induced depletion of cellular GSH in this cell line significantly reduced the formation of such conjugates from both drugs. However, concomitant increases in intracellular levels of reactive species were observed only after tetraplatin exposure. Our data suggest that the greater potentiation of PtIV‐ compared with PtII‐drug cytotoxicity in the relatively resistant cell lines following 24 hr BSO pre‐treatment may be caused by a differential effect of BSO on the metabolism and cellular distribution of these drugs. A BSO‐induced reduction in PtII‐ but not PtIV‐drug accumulation in these cells may also partially contribute to the differential potentiation of cytotoxicity of these drugs.
Immunization with interleukin‐2‐secreting allogeneic mouse fibroblasts expressing melanoma‐associated antigens prolongs the survival of mice with melanoma
Authors: Kim Tae Sung , Russell Stephen J. , Collins Mary K. L. , Cohen Edward P.
Published: February 2007

The survival of C57BL/6 mice (H‐2b) bearing B16 melanoma (H‐2b) was prolonged if the animals were treated solely by immunization with an interleukin‐2 (IL‐2)‐secreting allogeneic cell construct that expressed melanoma‐associated antigens (MAA) along with major histocompatibility complex (MHC) class‐l determinants (H‐2k; RLBA‐IL‐2 cells). This was the case if the mice were immunized simultaneously with, or 6 days following, the injection of viable B16 cells. Under similar conditions, the survival of tumor‐bearing mice immunized with an allogeneic cell construct (H‐2k) that expressed MAA but did not secrete IL‐2 (RLBA‐ZipNeo cells), or with an allogeneic construct (H‐2k) that secreted IL‐2 but did not form MAA (LM‐IL‐2 cells), was also prolonged. However, in these instances, the period of survival was significantly shorter than that of mice immunized with the cell construct that combined IL‐2 secretion with the expression of MAA. Tumor‐bearing mice immunized with non‐transfected LM(TK‐) cells (H‐2k), or irradiated B16 cells (H‐2b) failed to survive longer than untreated mice. Although the survival of the treated animals was prolonged, in most instances tumor growth recurred. The recurrent tumors in mice treated with the allogeneic cell constructs formed melanin and were histologically indistinguishable from tumors in untreated mice. Cells from the recurrent tumors were resistant to further immunotherapy and to cytotoxic effector cells obtained from the spleens of mice immunized with the same cellular immunogen used initially. The injection of IL‐2‐secreting syngeneic B16 cells into C57BL/6 mice invariably resulted in the appearance of non‐IL‐2‐secreting melanomas. Under similar circumstances, tumors failed to develop in C57BL/6 mice injected with IL‐2‐secreting, or non‐secreting, allogeneic cell constructs. Thus, the expression of allogeneic antigens protected the mice from growth of the cellular immunogens.
Estimates of the worldwide mortality from eighteen major cancers in 1985. Implications for prevention and projections of future burden
Authors: Pisani P. , Parkin D. M. , Ferlay J.
Published: February 2007

This report presents worldwide estimates of annual mortality from all cancers and for 18 specific cancer sites around 1985. Crude and age‐standardized mortality rates and numbers of deaths were computed for 24 geographical areas. Of the estimated 5 million deaths from cancer (excluding non‐melanoma skin cancer), 56% occurred in developing countries. The most frequent neoplasm is lung cancer, accounting for 22% of cancer deaths in men. Among women breast cancer is the leading malignancy, accounting for 16% and 11% of all cancer deaths in developed and developing countries, respectively. In developing countries, cancer of the cervix uteri ranks first, breast cancer second. The second most frequent cause of death from cancer in both sexes is cancer of the stomach, followed by liver cancer in men and by colon/rectum cancer in women. Cancers of the colon/rectum and prostate maintain a high rank in men living in developed countries, while cancers of the lung, ovary and pancreas occupy similar ranks among women. In developing countries, cancers of the oesophagus and mouth/pharynx follow those previously mentioned in both sexes. If the estimated rates continue to prevail, increases in the numbers of deaths of 20.4% in developed and 18.1 % in developing countries are expected by the year 2000, simply as a consequence of demographic trends towards ageing and population growth. Our results provide an indication of the potential impact of preventive practices. It is estimated that 20% of all cancer deaths (1 million) could be prevented by eliminating tobacco smoking. Mortality from cancers of the liver and uterine cervix, both major problems in developing countries, could be substantially reduced by immunization against hepatitis B virus infection and early detection through Pap smears, respectively.
Targeting of T lymphocytes against egf‐receptor+ tumor cells by bispecific monoclonal antibodies: Requirement of CD3 molecule cross‐linking for t‐cell activation
Authors: Ferrini Silvano , Cambiaggi Anna , Sforzini Sabrina , Marciano Sabrina , Canevari Silvana , Mezzanzanica Delia , Colnaghi Maria Ines , Grossi Carlo Enrico , Moretta Lorenzo
Published: February 2007

Targeting of T lymphocytes against epidermal growth‐factor‐receptor (EGF‐R)+ tumor cells was achieved by constructing a hybrid hybridoma which secretes an anti‐EGF‐R/anti‐CD3 bispecific monoclonal antibody (biMAb) of hybrid isotype (IgG1/ IgG2a). Purification of biMAb molecules from parental anti‐EGF‐R and anti‐CD3 MAbs was performed by protein‐A chromatography. The purified biMAb was able to trigger the lysis of EGF‐R+ tumor cell lines (A431, IGROV‐1, MDA‐468 and U‐87) and of NIH‐3T3 transfectants expressing human EGF‐R by cytolytic T lymphocytes, but it was ineffective in the case of EGF‐R‐negative tumor targets. Normal EGF‐R+ (keratinocytes and endometrial cells) were also susceptible to biMAb‐targeted cytolysis. However, the amount of biMAb required to induce half‐maximal cytolysis of tumor cells over‐expressing the EGF‐R molecule (A43I) was considerably lower than that required to induce lysis of EGF‐R+ tumor or normal cells which express EGF‐R at considerably lower density. The ability of such biMAbs to deliver activation signals to T cells was evaluated by Ca++ mobilization and lymphokine production experiments. The soluble anti‐EGF‐R/anti‐CD3 biMAb failed to induce intracellular Ca++ increases, which occurred only after cross‐linking induced by an anti‐mouse IgG antibody. Secretion of lymphokines (IFN‐γ, TNF‐α and GM‐CSF) was induced by contact of the biMAb‐coated effector cells with the relevant tumor target, whereas the soluble biMAb was virtually ineffective. In addition, biMAb‐coated effector cells retained the ability to recognize and to lyse EGF‐R+ tumor cells for a prolonged period of time. Our data indicate that activation of effector cells targeted by biMAbs can only occur at the tumor site, where cross‐linking of surface CD3 molecules is induced by contact with the tumor cells.
TPA‐enhanced invasion of matrigel associated with augmentation of cell motility but not metalloproteinase activity in a highly metastatic variant (L‐10) of human rectal adenocarcinoma cell line RCM‐1
Authors: Nabeshima Kazuki , Komada Naoto , Kishi Jun‐Ichi , Koita Hiroyuki , Inoue Teruhiko , Hayakawa Taro , Koono Masashi
Published: February 2007

We previously found that the enhanced activity to invade Matrigel upon stimulation with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was one of the major properties of a highly metastatic variant (L‐10) of a human rectal adenocarcinoma cell line RCM‐1. To clarify the mechanism of this enhancement, we examined the effect of TPA on 2 major biological factors involved in tumor cell invasion: cell motility and matrix‐degrading metalloproteinase activity. The enhanced invasiveness was inhibited by protein‐kinase‐C inhibitors. TPA markedly enhanced both haptotactic response to type‐IV collagen and motility on tissue‐culture glass substrate of L‐10 cells in a dose‐response manner quite similar to that of TPA‐enhanced invasion of Matrigel. On the other hand, TPA showed little enhancement of metalloproteinase production, which was assessed by gelatin‐ and casein‐zymography, and of type‐IV collagenolytic activity. Addition of TIMP (tissue inhibitors of metalloproteinase)‐1 inhibited TPA‐enhanced invasion of Matrigel by only up to 13%. Thus, TPA treatment of L‐10 cells enhanced invasion of Matrigel in association with augmentation of cell motility but did not enhance metalloproteinase activity.
Biochemical and immunological characterization of the human carcinoma‐associated antigen MH 99/KS 1/4
Authors: Schön Michael P. , Schön Margarete , Mattes M. Jules , Stein Rhona , Weber Lutz , Alberti Saverio , Klein C. Eberhard
Published: February 2007

We have characterized the 38‐kDa transformation‐associated membrane glycoprotein MH 99, whose expression is highly elevated in many epithelial malignancies. A spontaneous cleavage of MH 99 into a 32‐ and a 6‐kDa chain in some carcinoma cell lines was recently shown. Sequence homologies to nidogen, a matrix‐adhesion molecule, support the suggestion of a receptor‐like function. In this study, we characterized biochemical and immunogenic aspects of MH 99. Transformed epithelial cell lines which do not spontaneously cleave MH 99 were exposed to 8 proteases with distinct specificities. Each of the enzymes produced similar specific fragmentation into chains of about 30 to 32 and 6 kDa, indicating a characteristic cleavage site of MH 99. The fragments were not distinguishable from those in carcinoma cells showing spontaneous cleavage of MH 99. The specific fragmentation depends on the localization in intact membranes and is not shared by other membrane proteins. N‐glycosylation of MH 99 of about 4 kDa was exclusively found on the 32‐kDa fragment. Characterization of antigenic epitopes was performed using 16 different monoclonal antibodies (MAbs). Only 2 independent determinants were found. One is located on the 32‐kDa chain and is recognized only by the MM 104 MAb. The other 15 antibodies bind to a dominant epitope on the 6‐kDa fragment which can be divided into 3 overlapping sub‐epitopes. The unique features of MH 99 indicate that its immunogenic epitopes are mainly located at its 6‐kDa chain, and support the suggestion of a transformation‐associated cell‐surface receptor which might be proteolytically regulated.
Regulation of melanoma‐cell motility by the lipoxygenase metabolite 12‐(S)‐hete
Authors: Timar Jozsef , Silletti Steve , Bazaz Rajesh , Raz Avraham , Honn Kenneth V.
Published: February 2007

Cellular motility, a prerequisite for metastasis of tumor cells, is affected by a 55‐kDa tumor‐cell‐secreted cytokine which influences the migration of the producing cells and is called autocrine motility factor (AMF). Previous studies indicated that AMF stimulates motility by binding to its receptor, a cell‐surface glycoprotein of 78 kDa (gp78), inducing its phosphorylation, activating a pertussis toxin (PT)‐sensitive G‐protein, and stimulating inositol metabolism. However, the intracellular signaling mechanisms which transduce and regulate the AMF motility response remain largely unknown. 12‐(S)‐HETE, a lipoxygenase metabolite of arachidonic acid which affects the cytoskeletal architecture of murine melanoma cells, also stimulates cell motility independently of PT‐sensitive G‐proteins and up‐regulates gp78 surface expression. 12‐(S)‐HETE induces the phosphorylation of gp78 in a manner analogous to AMF and the motility response of these murine melanoma cells to both AMF and 12‐(S)‐HETE is inhibited by protein kinase C inhibitors. Furthermore, perturbation of the AMF receptor stimulated endogenous biosynthesis of 12(S)HETE. These results suggest the existence of an “autocrine motility cycle” which influences melanoma cell motility by gp78 activation, and production of second messengers which affect the cytoskeletal architecture and expression of the AMF receptor itself.
Ability of the non‐phorbol ester‐type tumor‐promoter thapsigargin to mimic the stimulatory effects of 12‐0‐tetradecanoylphorbol‐13‐acetate on ornithine decarboxylase activity, hydroperoxide production, and macromolecule synthesis in mouse epidermis In vivo
Authors: Perchellet Elisabeth M. , Gali Hala U. , Gao Xiao Mei , Perchellet Jean‐Pierre
Published: February 2007

The biochemical effects of the non‐12‐0‐tetradecanoylphorbol‐13‐acetate (TPA)‐type tumor promoter thapsigargin (TG), which does not bind to the phorbol‐ester receptor, or activate protein kinase C (PKC) or increase inositol polyphosphates, were characterized in mouse epidermis in vivo. The cold scraping method is required to detect the induction of ornithine decarboxylase (ODC) activity by TG, a response much smaller than that caused by TPA and with a different time course, TG pre‐treatments do not alter or cause a refractory state against ODC induction by TPA. But TG stimulates hydroperoxide (HPx) production and RNA, protein, and DNA synthesis almost as much as TPA. Moreover, the sequential effects of TG and TPA on DNA synthesis are identical: early inhibition at 8 hr followed by maximal stimulation at 16‐32 hr. TG‐stimulated HPx production requires protein synthesis and xanthine oxidase, phospholipase A2, and lipoxygenase activities but not RNA and DNA synthesis, and cyclooxygenase and protease activities. The HPx response to TG is not mimicked by the PKC activator prostratin or inhibited by pre‐treatments with prostratin or specific PKC inhibitors. However, the Ca2+‐ATPase inhibitor cyclopiazonic acid and the Ca2+ ionophore and weak ODC inducer A23187 mimic remarkably the HPx responses to TG and TPA. Since TG and A23187 are known to be, respectively, weak and incomplete tumor promoters as compared with TPA, the present results suggest that the HPx responses common to Ca2+‐mobilizing and TPA‐ or non‐TPA‐type agents are insufficient to achieve tumor promotion in the absence of major ODC induction.
High‐fiber diets and reduced risk of breast cancer
Authors: Baghurst Peter A. , Rohan Thomas E.
Published: February 2007

In a case‐control study of 451 women with breast cancer and 451 population‐based controls from metropolitan Adelaide, Australia, the risk of breast cancer was studied in relation to intake of dietary fiber and various fiber components. Intakes were estimated by means of a self‐administered quantitative food‐frequency questionnaire, and risks of breast cancer were estimated for each quintile of fiber density (intake/megajoule of total energy intake) relative to an arbitrarily assigned risk of unity for women in the lowest quintile of fiber density. There were highly significant reductions in relative risk associated with increasing intake of a number of components of total dietary fiber. In women with the highest 20% estimated dietary densities of total non‐starch polysaccharides (NSP), the relative risk of breast cancer was 0.46, and a test for trend across the quintiles was significant at p < 0.001. Separate risk estimations for various fiber components defined by the Enlists method of analysis revealed highest risk reductions associated with high densities of mannose from insoluble NSP and of glucose from soluble NSP. For many fiber components the point estimates of relative risk were quite similar both for pre‐and for postmenopausal women, although results for the latter (who comprised over two thirds of all cases) were statistically more significant. The study provides strong support for recent conjecture that foods rich in dietary fiber may be protective against breast cancer.
Identification of integrated epstein‐barr virus in nasopharyngeal carcinoma using pulse field gel electrophoresis
Authors: Kripalani‐Joshi S. , Law H. Y.
Published: February 2007

Integrated Epstein‐Barr virus (EBV) was found in 4 of 17 pathologically diagnosed, EBV‐positive nasopharyngeal carcinoma (NPC) biopsy samples using pulse field gel electrophoresis (PFGE). The analyses were carried out after digestion of biopsy DNA with rare restriction enzymes such as Pacl, which does not cleave EBV due to the absence of the recognition sequence, and Clal. Employing this technique, EBV integration in IB4, through Ecol, and Namalwa through the terminal repeats, was confirmed. We also established that integration of EBV in AW Ramos was through the terminal repeats. The Raji cell line was also found to harbour integrated virus in addition to episomal DNA. However, the site of integration could not be confirmed, since as the Raji DNA appeared to be heavily methylated and could not be cleaved with CPG rare cutters. As in the Raji cell line, the DNA of EBV in nasopharyngeal‐carcinoma (NPC) biopsies also appeared to be methylated.
Genetic alteration of the hepatitis C virus hypervariable region obtained from an asymptomatic carrier
Authors: Nakazawa Takahide , Kato Nobuyuki , Ootsuyama Yuko , Sekiya Hitomi , Fujioka Takahiro , Shibuya Akitaka , Shimotohno Kunitada
Published: February 2007

Hepatitis C virus (HCV) genome shows extensive sequence diversity at 2 hypervariable regions (HVR1 and HVR2) of the putative envelope glycoprotein (gp70). We recently reported that the amino‐acid sequence of HVR1, but not of HVR2, underwent a striking mutation or mutated sequentially over a period of several months in patients with chronic hepatitis (CH). Here, we examined whether these genetic alterations in HVR1 occurred in an asymptomatic HCV carrier. The level of HCV RNA in serum was almost the same throughout the 4 time points sampled over 16 months. However, we found that the amino‐acid sequence of the HCV HVR1 from this asymptomatic carrier altered with time, as seen in patients with CH. Alterations of amino acids in the HVR1 were correlated with persistent HCV infection rather than with clinical symptoms. Sequence heterogeneity of HVR1 was not correlated with alanine aminotransferase (ALT) values or liver histological findings. The necessity of clinical follow‐up of HCV asymptomatic carriers is discussed.
Induction of tissue‐type plasminogen activator and 72‐kDa type‐IV collagenase by ionizing radiation in rat astrocytes
Authors: Sawaya Raymond , Tofion Philip J. , Mohanam Sanjeeva , Ali‐Oosman Francis , Liotta Lance A. , Stetler‐Stevenson William G. , Rao Jasti S.
Published: February 2007

Radiation‐induced damage in the central nervous system (CNS) is believed to be targeted to glial or endothelial cells or both, although the pathophysiology of the process is still poorly understood. In this study, we irradiated rat astrocytes with single doses of X‐rays and then estimated the levels of tissue plasminogen activator (tPA) and collagenase in serum‐free medium and cell extracts at different times. Fibrin zymography revealed increased levels of intracellular tPA activity at 12 hr after irradiation. Gelatin zymography showed continuously increasing levels of extracellular 72‐kDa type‐IV collagenase after irradiation. Quantitative enzymatic activities by densitometry showed a 3‐to 4‐fold elevation in the level of the intracellular tPA activity at 12 hr and a 5‐to 6‐fold increase in the level of the extracellular 72‐kDa type‐IV collagenase activity at 48 hr. An ELISA with specific antibodies for tPA and 72‐kDa type‐IV collagenase indicated a 5‐fold increase in the level of tPA at 12 hr and a more‐than‐7‐fold increase in the level of 72‐kDa type‐IV collagenase at 48 hr. This study adds considerable credibility to the proposed role of plasminogen activators and type‐IV collagenase in the development of CNS damage after radiotherapy for brain tumors.
Growth inhibition of human melanoma‐derived cells by 12‐O‐tetradecanoyl phorbol 13‐acetate
Authors: Arita Yuko , O'Driscoll Kevin R. , Weinstein I. Bernard
Published: February 2007

In vitro growth of 6 human melanoma‐derived cell lines was inhibited markedly by the phorbol‐ester tumor promoter 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA), a potent activator of several isoforms of protein kinase C (PKC). Utilizing PKC isoform‐specific antibodies in immunoblotting experiments, we found that the PKCα and PKC isoforms were expressed in all of the 6 melanoma cell lines tested, whereas the PKCβ isoform was expressed at detectable levels in only 2 of the 6 cell lines. The SK‐Mel‐173 melanoma cell line, which had relatively high levels of PKCβ mRNA and protein expression, and which was also the most sensitive to cell growth inhibition by TPA, was used to isolate clones whose growth was less inhibited by TPA. Immunoblotting experiments revealed that in parental SK‐Mel 173 cells PKCβ was rapidly down‐regulated to below detectable levels after treatment for 48 hr with TPA, but that in TPA‐resistant variant clones there was negligible down‐regulation of PKCβ by TPA. On the other hand, treatment of parental and TPA‐resistant SK‐Mel 173 cells with TPA led to partial down‐regulation of PKCα in both cell lines. Total PKC enzyme activity was also greater in TPA‐resistant cells than in parental SK‐Mel 173 cells. Our results show that TPA might inhibit the growth of melanoma cells by causing down‐regulation of specific isoforms of PKC that are required to maintain the growth of these cells.
Heterogeneous response to the growth factors [EGF, PDGF (bb), TGF‐α, bFGF, IL‐2] on glioma spheroid growth, migration and invasion
Authors: Pedersen Paal‐Henning , Ness Gro Oddveig , Engebraaten Olav , Bjerkvig Rolf , Lillehaug Johan R. , Laerum Ole Didrik
Published: February 2007

The effects of 5 different growth factors [EGF, PDGF(bb), TGF‐α, bFGF and IL‐2] were studied on tumour spheroids obtained from 5 different human glioma cell lines (U‐251MG, D‐263MG, D‐37MG, D‐54MG, GaMG). The expression of EGF and PDGF receptors as well as the endogenous production of TGF‐α and PDGF Were studied by Northern blot analyses. After growth‐factor‐exposure, tumour spheroid volume growth, and directional cell migration from the spheroids were studied. In addition, tumour‐cell invasion was studied In vitro, where foetal rat‐brain aggregates were used as a target for the tumour cells. In all the assays a common stimulator for most of the cell lines was EGF. The other growth factors had a more heterogeneous stimulatory effect. Tumour‐cell invasion, cell growth and cell migration are biological properties which are not necessarily related to each other. This may explain why the tumours often responded differently to the growth factors in the various assay systems. Two of the cell lines studied were non‐invasive (U‐25IMG, D‐263MG). It is shown that these were stimulated both in the directional migration assay and in the spheroid‐volume‐growth assay. However, their non‐invasive behaviour was not influenced by the growth factors studied.
Therapeutic effects of monoclonal antibody g250, interferons and tumor necrosis factor, in mice with renal‐cell carcinoma xenografts
Authors: van Dijk J. , Uemura H. , Beniers A. J. M. C. , Peelen W. P. , Zegveld S. Th. , Fleuren G. J. , Warnaar S. O. , Oosterwijk E.
Published: February 2007

Because renal‐cell carcinoma (RCC) is considered relatively resistant to radio‐and chemotherapy, RCC patients may benefit from new treatment modalities, e.g. immunotherapy. In vitro and in vivo studies suggest that combinations of cytokines such as interferon γ or interferon a (IFN‐γ, IFN‐α) and tumor necrosis factor a (TNF‐α) may act synergistically. In this study we tested whether a monoclonal antibody (MAb) G250, reactive with a RCC‐associated antigen, showed anti‐tumor effects in vivoin nude mice with established s.c. human RCC xenografts, and also whether this MAb could enhance the anti‐tumor effect of combinations of IFNs and TNF‐α. Treatment with combinations of IFN‐α/TNF‐α or IFN‐γ/TNF‐α, or with MAb G250 alone, resulted in a significant inhibition of tumor growth. Treatment with MAb G250, in combination with IFN‐γ/TNF‐α, did not result in an improve anti‐tumor effect as compared to that of either treatment alone. In contrast, MAb G250 combined with IFN‐α/TNF‐α resulted in a significantly enhanced anti‐tumor response. In one experiment, 3 out of 10 mice showed complete tumor regression, with no recurrence after 90 days. Large numbers of infiltrating macrophages were found surrounding viable and necrotic tumor tissue after treatment with G250 combined with IFN‐α/TNF‐α. These results suggest that combination therapy, consisting of IFN‐α, TNF‐α and MAbs, may have therapeutic value in the treatment of RCC.
Resistance to the anti‐proliferative effect of IL‐1 on human melanoma cell line is associated with endogenous production of IL‐1 and IL‐6
Authors: Araki Makoto , Yano Taishi , Hayashi Hidetoshi , Takii Takemasa , Suzuki Katsuya , Onozaki Kikuo
Published: February 2007

A human melanoma cell line (A375–6) became resistant to the anti‐proliferative effect of human IL‐I after a long period of culture. Two stable resistant sub‐clones were obtained, and the mechanism of the IL‐I resistance was investigated. Resistant cells, but not sensitive cells, appeared to produce constitutiveiy IL‐I activity. The activity was neutralized by anti‐IL‐I a antibody but not by anti‐IL‐I β antibody. Resistant cells expressed IL‐I α but not IL‐Iβ mRNA. Therefore, the resistant cells appeared to produce IL‐β α. mRNA for IL‐I receptor antagonist (IL‐I Ra) was not detected in resistant cells, indicating that the resistance is not attributable to IL‐IRa. These resistant cells were also resistant to the anti‐proliferative effect of human IL‐6, but not to that of human TNF. Resistant cells appeared to produce constitutively IL‐6 more than sensitive cells, and IL‐6 production both in sensitive and in resistant cells was augmented by exogenous IL‐I. Furthermore, constitutive production of IL‐6 in resistant cells was inhibited by IL‐I Ra. Type I IL‐I receptor (IL‐IR) mRNA was expressed equally in resistant and sensitive cells. These data indicate that the resistance is not the result of loss of functional IL‐IR and that IL‐I induces IL‐6 in an autocrine manner. It is, therefore, conceivable that endogenous IL‐I and IL‐6 contribute to IL‐I resistance.