bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemoprotective effect of caffeic acid phenethyl ester on promotion in a medium‐term rat hepatocarcinogenesis assay
Authors: Carrasco‐Legleu Claudia E. , Márquez‐Rosado Lucrecia , Fattel‐Fazenda Samia , Arce‐Popoca Evelia , Pérez‐Carreón Julio I. , Villa‐Treviño Saúl
Published: June 2004

Caffeic acid phenethyl ester (CAPE), a natural honeybee product exhibits a spectrum of biological activities including anti‐microbial, anti‐inflammatory, antioxidant and anti‐tumoral actions. CAPE is also chemopreventive against intestinal, colon and skin cancer. Our aim was to extend the study of its chemoprotective features to the promotion of hepatocarcinogenesis. Male Wistar rats were subjected to a protocol under a modified promotion regimen of the resistant hepatocyte model. The altered hepatic foci (AHF) were quantitatively analyzed by histochemistry and image processing. When given during promotion, CAPE (20 mg/kg) decreased the expression of number and area γ‐glutamyl transpeptidase (GGT) positive AHF by 91% and 97%, respectively. When GGT expression was analyzed by RT‐PCR, CAPE drastically decreased and prevented expression of almost all GGT transcripts at this stage of the carcinogenic process. Glutathione S‐transferase placental form (GST‐P), another protein marker for preneoplastic lesions was measured by Western blot and a decrease of 82% was observed. Additionally, we evaluated the effect of CAPE on the expression of nuclear factor NF‐κB and found an 85% decrease in nuclear localization of the p65 subunit of NF‐κB; however, their repressor, IκBα was not modified. Our results showed that CAPE given during promotion in hepatocarcinogenesis protects against induction of GGT‐positive AHF, GST‐P protein, GGT mRNA expression and translocation of p65. This phenomenon was independent of IκBα degradation. © 2003 Wiley‐Liss, Inc.
Childhood leukemia and road traffic: A population‐based case‐control study
Authors: Crosignani Paolo , Tittarelli Andrea , Borgini Alessandro , Codazzi Tiziana , Rovelli Adriano , Porro Emma , Contiero Paolo , Bianchi Nadia , Tagliabue Giovanna , Fissi Rosaria , Rossitto Francesco , Berrino Franco
Published: June 2004

To assess the effect of road traffic exhaust on the risk of childhood leukemia, we carried out a population‐based case‐control study in the Province of Varese, northern Italy, covered by a population‐based cancer registry. All 120 incident cases from 1978–97 were included in the study. Four controls per case, matched by age and gender, were sampled from population files. As index of exposure to traffic exhaust we estimated the annual mean concentration of benzene outside the home using a Gaussian diffusion model. This model uses traffic density (vehicles/day) on nearby main roads, distance between roads and residence, and information on vehicle emissions and weather conditions to estimate benzene concentration. Compared to children whose homes was not exposed to road traffic emissions (<0.1 μg/m3 of benzene as estimated by the model), the risk of childhood leukemia was significantly higher (relative risk [RR] = 3.91; 95% confidence interval [CI] = 1.36–11.27) for heavily exposed children (over 10 μg/m3 estimated annual average). For the intermediate exposure group (0.1–10 μg/m3) the relative risk was 1.51 (95% CI = 0.91–2.51). These data, considered with other available evidence, suggest that motor traffic emissions can be involved in the etiology of childhood leukemia. © 2003 Wiley‐Liss, Inc.
Menstrual and reproductive factors and endometrial cancer risk: Results from a population‐based case‐control study in urban Shanghai
Authors: Xu Wang‐Hong , Xiang Yong‐Bing , Ruan Zhi‐Xian , Zheng Wei , Cheng Jia‐Rong , Dai Qi , Gao Yu‐Tang , Shu Xiao‐Ou
Published: June 2004

The purpose of our study was to evaluate the association of menstrual and reproductive factors with the risk of endometrial cancer. In a population‐based case‐control study conducted in urban Shanghai, in‐person interviews were completed for 833 women aged 30–69 years and an equal number of controls frequency‐matched to cases by age. All cases were newly diagnosed with endometrial cancer between January 1, 1997 and December 31, 2001. The unconditional logistic regression model was employed to derive the adjusted odds ratios (ORs) of endometrial cancer and 95% confidence intervals (CIs) in relation to menstrual and reproductive factors. Earlier menarche age, particularly among premenopausal women, and later menopausal age were associated with an elevated risk of endometrial cancer. A clear dose‐response relation between endometrial cancer risk and years of menstruation was observed (p for trend < 0.01). Compared to women ever having a pregnancy and women ever having had a live birth, respectively, nulligravity and nulliparity were both associated with a more than one‐fold elevated risk of endometrial cancer. Both completed (OR = 3.02, 95% CI 1.10–8.32 for women never having a complete pregnancy) and incomplete pregnancy (OR = 0.69, 95%CI 0.55–0.87) conferred a protective effect against endometrial cancer, and the protective effect appeared to increase with total number of pregnancies (p for trend = 0.01). The effect of pregnancy on endometrial cancer remained unchanged with increasing time since the last pregnancy. Stillbirth and age at first pregnancy was unrelated to endometrial cancer risk. Our study suggests that prolonged menstruation was related to an increased risk of endometrial cancer while pregnancy, including induced abortion, reduced the risk of endometrial cancer. © 2003 Wiley‐Liss, Inc.
HER‐2 receptor expression, localization, and activation in colorectal cancer cell lines and human tumors
Authors: Half Elizabeth , Broaddus Russell , Danenberg Kathleen D. , Danenberg Peter V. , Ayers Gregory D. , Sinicrope Frank A.
Published: June 2004

The HER‐2/neu oncogene encodes a 185 kD protein that is phosphorylated upon ligand binding to other HER/erbB members and regulates cell growth and differentiation. Given that HER‐2 receptor blockade can inhibit the growth of colon cancer cell lines and tumor xenografts, we investigated the frequency, localization and phosphorylation status of HER‐2 in colon cancer cell lines and in human tumors. Protein expression was analyzed in relation to mRNA levels, HER‐2 amplification, and clinicopathological variables. Colon cancer cell lines constitutively expressed HER‐2 proteins and none showed HER‐2 amplification by fluorescence in situ hybridization. Cell fractionation and immunoblotting showed HER‐2 in both the membrane and cytosolic compartments. Primary colorectal carcinomas (n = 96) and their metastases (n = 25) were examined by immunohistochemistry. Strong membrane HER‐2 staining was detected in 5 (5%) of primaries and in 3 (12%) metastases (p = 0.36). Membrane but not cytoplasmic localization was strongly associated with HER‐2 gene amplification (p = 0.007). Cytoplasmic HER‐2 staining was found in 61 (63.5%) of primary tumors and localization was confirmed by immunoelectron microscopy that also showed plasma membrane HER‐2. Using real‐time quantitative RT‐PCR, HER‐2 mRNA was increased in tumors with membrane compared to cytoplasmic staining (r = 0.66, p = 0.001). Cytoplasmic HER‐2 was associated with tumor differentiation (p = 0.018), but not other clinicopathological variables. By immunoblotting, heterogeneity was seen in HER‐2 levels with downregulation in 4 of 7 tumors relative to normal epithelia that uniformly expressed HER‐2. Phosphorylated HER‐2 was detected in approximately 50% of tumors and in normal mucosa. In conclusion, HER‐2 is expressed constitutively in colon cancer cell lines and demonstrates relatively distinct localization patterns in human tumors. Strong membrane immunoreactivity is associated with high levels of HER‐2 mRNA and gene amplification whereas cytoplasmic HER‐2 is detected frequently and seems to be a marker of tumor differentiation. © 2003 Wiley‐Liss, Inc.
Partial tyrosinase‐specific self tolerance by HLA‐A*0201‐restricted cytotoxic T lymphocytes in mice and man
Authors: Lotz Carina , Ferreira Edite Antunes , Drexler Ingo , Mutallib Sarah Abdel , Huber Christoph , Sutter Gerd , Theobald Matthias
Published: June 2004

The human tyrosinase (hTyr) (369–377) cytotoxic T lymphocyte (CTL) epitope is presented by malignant melanoma and various nontransformed cells in association with human leukocyte antigen (HLA)‐A*0201 (A2.1) and used for vaccination‐based immunotherapy of melanoma patients. Its mouse homologue, mTyr (369‐377), is naturally processed and bound by A2.1 with equivalent efficacy and thus enabled us to explore the effect of self tolerance on Tyr‐specific T cells in different lines of A2.1 transgenic (Tg) mice and man. We found that self Tyr‐reactive CTL in Tg mice and, importantly, in man were affected by partial tolerance resulting in only residual T lymphocytes of higher avidity for self Tyr along with low‐avidity T cells to be present in the periphery. Immunizing mice with the xenogeneic nonself Tyr peptide facilitated the generation of self Tyr‐reactive CTL. As compared to Tyr‐reactive CTL induced by high amounts of the self Tyr epitope, however, the nonself antigen (Ag) had no effect on improving the avidity of self Tyr‐specific mouse and human T cells. Depleting mice of CD25+ T cells with and without CTL‐associated Ag 4 (CTLA‐4) blockade demonstrated that tolerance of Tyr‐specific CTL was not regulated by CD4+CD25+ T regulatory cells (Treg) or CTLA‐4. Our studies have important implications for the design of anti‐Tyr‐based immunotherapeutics. © 2003 Wiley‐Liss, Inc.
Decreased pyruvate kinase M2 activity linked to cisplatin resistance in human gastric carcinoma cell lines
Authors: Yoo Byong Chul , Ku Ja‐Lok , Hong Sung‐Hye , Shin Young‐Kyoung , Park So Yeon , Kim Hark Kyun , Park Jae‐Gahb
Published: June 2004

Resistance to anticancer drugs is a major obstacle preventing effective treatment of disseminated cancers. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cisplatin or 5‐fluorouracil (5‐FU) were established from human gastric carcinoma cell lines SNU‐638 and SNU‐620. Comparative proteomics involving 2‐dimensional gel electrophoresis (2‐DE) and matrix‐associated laser desorption ionization‐mass spectroscopy (MALDI‐MS) was performed on protein extracts from these parental and drug‐resistant derivative lines to screen drug resistance‐related proteins. Pyruvate kinase M2 (PK‐M2) was identified as a protein showing lower expression in cisplatin‐resistant cells compared to parental cells. Consistent with this finding, PK‐M2 activity was also lower in cisplatin‐resistant cells. Suppression of PK‐M2 expression by antisense oligonucleotide resulted in acquired cisplatin resistance in SNU‐638 cells. Furthermore, PK‐M2 activity in 11 individual human gastric carcinoma cell lines positively correlated with cisplatin sensitivity. Taken together, PK‐M2 protein and activity levels were lower in cisplatin‐resistant human gastric carcinoma cell lines compared to their parental cell lines. Furthermore, suppression of PK‐M2 expression using antisense oligonucleotides increased cisplatin resistance. These data clearly link PK‐M2 and cisplatin resistance mechanisms. © 2003 Wiley‐Liss, Inc.
Association of kallikrein expression in nipple aspirate fluid with breast cancer risk
Authors: Sauter Edward R. , Lininger John , Magklara Angelika , Hewett John E. , Diamandis Eleftherios P.
Published: June 2004

Human kallikreins (hK) 2, 3, 6 and 10 are expressed in breast and prostate tissue. hK2 and hK3 (prostate‐specific antigen, PSA) are used to screen for prostate cancer. hK6 and hK10 are downregulated in breast cancer compared to normal breast tissue. We demonstrated that levels of PSA in nipple aspirate fluid (NAF) are lower in women with breast cancer than in normal women. We hypothesize that the expression of hK2, 3, 6 and 10 are related and important in detecting breast cancer. The goals of this study are to determine the level of expression of kallikreins in NAF and serum, the association of hK2, 3, 6 and 10 in NAF, and the association of each of the kallikreins with breast cancer. In NAF from 275 women, hK3, 6 and 10 were detectable in ≥ 90% and hK2 in 74% of samples analyzed. NAF levels were highest for hK6 and lowest for hK2, regardless of cancer and menopausal status. hK3 was detectable in 15/29 (52%) and hK2 in 0/29 serum samples collected from 6 women. hK2 and hK3 were concentrated in NAF vs. matched serum. The 4 kallikreins were associated with the exception of hK2 with hK6 or hK10. PSA levels were higher in normal pre‐ than postmenopausal subjects (but not women with breast cancer), whereas levels of hK2, 6 and 10 did not differ by menopausal status. hK2 and PSA were associated with both pre‐ and postmenopausal breast cancer; hK6 and 10 were not. hK2 and PSA were more associated with pre‐ than postmenopausal breast cancer. Using logistic regression, PSA and menopausal status provided the best model of breast cancer prediction, with a sensitivity of 91% and specificity of 39%. In conclusion, 4 kallikreins are expressed in NAF. hK2 and PSA, and hK6 and hK10 are highly associated. Higher premenopausal PSA levels suggest the influence of ovarian steroids. PSA shows the most promise in aiding in the early detection of breast cancer. © 2003 Wiley‐Liss, Inc.
Generation, immunologic characterization and antitumor effects of human monoclonal antibodies for carcinoembryonic antigen
Authors: Imakiire Takayuki , Kuroki Motomu , Shibaguchi Hirotomo , Abe Hironori , Yamauchi Yasushi , Ueno Aruto , Hirose Yumiko , Yamada Hiromi , Yamashita Yuichi , Shirakusa Takayuki , Ishida Isao , Kuroki Masahide
Published: June 2004

We generated fully human mAbs (HmAbs) to carcinoembryonic antigen (CEA) using the KM mouse, which carries a human chromosome 14 fragment containing the entire Ig H chain loci and human κ L chain segments in the mouse genome. Forty‐six hybridoma clones producing HmAbs to CEA were thus obtained by fusing the P3‐U1 mouse myeloma cells with splenocytes of the KM mice immunized with CEA. Among them, 22 clones produced HmAbs that reacted with CEA but not with 3 other CEA‐related cell adhesion molecule (CEACAM) family members, CEACAM1, CEACAM6 and CEACAM8. In 12 HmAbs examined, 8 were IgG4, 2 were IgG3, 1 was IgG2, and the other was IgG1. The affinity constants for CEA of these HmAbs were comparable to those of the previously prepared mouse anti‐CEA mAbs (MmAbs). BIAcore analyses revealed that 1 and 2 of the 22 HmAbs react with 2 epitopes defined by MmAbs on the domain N and the domain A1 or B1 of CEA, respectively. In the presence of human complement in vitro, 2 HmAbs tested showed substantial cytotoxicity, namely, 50–65%, against CEA‐expressing tumor cells. With human lymphokine‐activated killer cells in vitro, 3 HmAbs tested exhibited 40–65% Ab‐dependent cell‐mediated cytotoxicity against the tumor cells. Moreover, one of the HmAbs induced a significant inhibition of tumor growth when administered to mice xenografted with the CEA‐expressing cells. Considering their lack of immunogenicity to humans, these CEA‐specific HmAbs may be useful for immunotherapeutic approaches as well as for immunodiagnosis. © 2003 Wiley‐Liss, Inc.
Highly sensitive detection of the MGB1 transcript (mammaglobin) in the peripheral blood of breast cancer patients
Authors: Cerveira Nuno , Torres Lurdes , Rocha Patrícia , Bizarro Susana , Pereira Deolinda , Abreu Joaquim , Henrique Rui , Teixeira Manuel R. , Castedo Sérgio
Published: June 2004

We describe a new one‐step RT‐PCR assay for the detection of the mammaglobin (MGB1) gene transcript in the peripheral blood of breast cancer patients. With this approach, the MGB1 transcript could be detected in the peripheral blood of 22 of 54 (41%) breast cancer patients prior to any therapy. This method, using specific primers for cDNA synthesis, proved to be more sensitive (10−6 to 10−11, usually 10−7) than previously reported methodologies. This increased sensitivity was achieved without compromising specificity, as the MGB1 transcript was not detected in 38 blood samples of healthy donors and in only 1 of 18 blood samples of patients presenting with hematologic malignancies. A positive correlation was seen between MGB1 positivity and breast cancer stage: 0/3 (0%) in stage 0, 3/13 (23%) in stage I, 6/17 (35%) in stage II, 5/10 (50%) in stage III, 8/11 (73%) in stage IV (p = 0.003). The prognostic and therapeutic implications of MGB1 positivity by one‐step RT‐PCR in the peripheral blood of breast cancer patients, especially in clinically localized disease (stages I and II), should be evaluated after long‐term clinical follow‐up of these patients. © 2003 Wiley‐Liss, Inc.
The efficacy of ritonavir in the prevention of AIDS‐related Kaposi's sarcoma
Authors: Stebbing Justin , Portsmouth Simon , Nelson Mark , Mandalia Sundhiya , Kandil Hala , Alexander Nic , Davies Liz , Brock Cathryn , Bower Mark , Gazzard Brian
Published: June 2004

Angiogenesis is thought to play a major role in the development of Kaposi's sarcoma (KS), considered by many to be a hyperplastic disorder caused in part by local production of inflammatory cytokines. The antiangiogenic effects of protease inhibitors, in particular ritonavir, have been suggested in laboratory work to lead to regression of KS, and recent data have shown the importance of ritonavir as a model of pharmaceutical development. As our clinical cohort data has shown that non‐nucleoside reverse transcriptase inhibitor‐based regimens are not inferior to protease inhibitor‐based therapy in the prevention of KS, we investigated the specific contribution of ritonavir to chemoprevention of this AIDS‐defining illness. In a logistic regression analysis, we found that ritonavir‐based therapy confers no advantages compared to other regimens in the prevention of KS. This is consistent with data suggesting that regression of KS is mediated by an overall improvement in immune function and not by the effects of specific antiretrovirals. © 2003 Wiley‐Liss, Inc.
Inhibition of human vascular endothelial cells proliferation by terbinafine
Authors: Ho Pei‐Yin , Liang Yu‐Chih , Ho Yuan‐Soon , Chen Chiung‐Tong , Lee Wen‐Sen
Published: June 2004

We have demonstrated previously that terbinafine (TB), an oral antifungal agent used in the treatment of superficial mycosis, suppresses proliferation of various cultured human cancer cells in vitro and in vivo by inhibiting DNA synthesis and activating apoptosis. In our study, we further demonstrated that TB at a range of concentrations (0–120 μM) dose‐dependently decreased cell number in cultured human umbilical vascular endothelial cells (HUVEC). Terbinafine was not cytotoxic at a concentration of 120 μM, indicating that it may have an inhibitory effect on the cell proliferation in HUVEC. The TB‐induced inhibition of cell growth rate is reversible. [3H]thymidine incorporation revealed that TB reduced the [3H]thymidine incorporation into HUVEC during the S‐phase of the cell‐cycle. Western blot analysis demonstrated that the protein levels of cyclin A, but not cyclins B, D1, D3, E, CDK2 and CDK4, decreased after TB treatment. The TB‐induced cell‐cycle arrest in HUVEC occurred when the cyclin‐dependent kinase 2 (CDK2) activity was inhibited just as the protein level of p21 was increased and cyclin A was decreased. Pretreatment of HUVEC with a p21 specific antisense oligonucleotide reversed the TB‐induced inhibition of [3H]thymidine incorporation. Taken together, these results suggest an involvement of the p21‐associated signaling pathway in the TB‐induced antiproliferation in HUVEC. Capillary‐like tube formation and chick embryo chorioallantoic membrane (CAM) assays further demonstrated the anti‐angiogenic effect of TB. These findings demonstrate for the first time that TB can inhibit the angiogenesis. © 2004 Wiley‐Liss, Inc.
Low urine osmolarity as a determinant of cisplatin‐induced nephrotoxicity
Authors: Polycarpe Emmanuel , Arnould Laurent , Schmitt Elise , Duvillard Laurence , Ferrant Emmanuelle , Isambert Nicolas , Duvillard Christian , Beltramo Jean‐Luc , Chevet Dominique , Chauffert Bruno
Published: June 2004

Cisplatin is widely used in the treatment of human tumors, but it is a nephrotoxic drug. Early pragmatic clinical trials have shown that cisplatin‐induced renal toxicity is greatly reduced through the use of high hydration, a large NaCl supply and mannitol infusion, but the precise mechanisms of these nephroprotective measures are not fully understood. We show here an increase in the cisplatin uptake and cytotoxicity on 56/10 A1 human glomerular and HK‐2 human tubular cells when the drug incubation was performed in a hypotonic phosphate‐buffered saline solution or in human urine (“drag in” transport hypothesis). When 4 mg/kg cisplatin was intraperitoneally injected in rats in 20 ml of a hypotonic 4 g/l NaCl solution, the platinum accumulation increased in both the cortex and papilla but not in the subcutaneously grafted colon tumors when compared to rats injected with cisplatin in normal or hyperosmotic solutions (9 and 14 g/l NaCl, respectively). The urea and creatinine blood levels were significantly increased, and more apoptotic cells were detected by the caspase‐3 cleavage and TUNEL assays in the tubular cells of rats treated with cisplatin in a hypotonic solution compared to animals that received normal or hypertonic solutions. Osmolarity was sometimes low in urine from patients receiving an intravenous hydration for a cisplatin treatment or from healthy volunteers who were given an oral hydration with a 50 g/l glucose solution. Our results show that low urine osmolarity could be a major determinant in the increase of cisplatin‐induced nephrotoxicity and justify the widely used concurrent infusion of osmotically active substances during intravenous hydration. © 2004 Wiley‐Liss, Inc.
Glycine‐extended gastrin induces matrix metalloproteinase‐1‐ and ‐3‐mediated invasion of human colon cancer cells through type I collagen gel and Matrigel
Authors: Baba Miyako , Itoh Kazuyuki , Tatsuta Masaharu
Published: June 2004

The effects of glycine‐extended gastrin (G‐Gly) on the invasion by colon cancer cells through stromal extracellular matrix and the role of metalloproteinases (MMPs) in this invasion were investigated. We found that 10−9–10−6 M G‐Gly significantly increased the invasiveness of 2 human colon cancer cell lines, LoVo and HT‐29, both expressing the G‐Gly‐specific binding site but little gastrin/CCK‐B receptor (gastrin receptor). LoVo cells expressed MMP‐1, ‐2, ‐3 and ‐9. An amount of 10−7 M G‐Gly enhanced collagenase MMP‐1 expression. Overexpression of enhanced green fluorescent protein (EGFP)‐fused MMP‐1 in LoVo cells, by cDNA transfection, enhanced invasiveness through type I collagen gel. Immunofluorescence study revealed that G‐Gly increased the number of cytoplasmic vesicles containing MMP‐1, some vesicles being released from the cells. The MMP‐1 vesicles contained one of the ubiquitous coat proteins, Golgi‐localized, γ‐adaptin ear‐containing, ARF‐binding proteins‐2 (GGA‐2). MMP‐1 also colocalized with CD147 (EMMPRIN, an extracellular matrix metalloproteinase inducer in adjacent stromal cells). It was suggested that G‐Gly increased the number of vesicles containing MMP‐1 and that MMP‐1 interacted with CD147 to increase invasion. G‐Gly significantly enhanced the production of MMP‐3, an activator of MMP‐1 and ‐9, as well as gelatinase MMP‐9 activity. The G‐Gly‐mediated MMP‐9 increase was inhibited by treatment with anti‐MMP‐3 IgG and MMP‐3 siRNA. Furthermore, G‐Gly increased the proMMP‐2 level, although no activated MMP‐2 was found in conditioned medium in either the presence or the absence of G‐Gly. By contrast, gastrin (10−7 M) had no effect on the levels of these MMPs or the invasiveness of colon cancer cells in type I collagen gel and Matrigel. These effects of G‐Gly on the activity and expression of MMPs and the invasiveness of colon cancer cells were inhibited by treating the cells with a broad‐spectrum metalloproteinase inhibitor (CGS27023A) and nonselective gastrin/CCK receptor antagonists (proglumide and benzotript). But a gastrin/CCK‐B receptor antagonist (YM022) did not inhibit the increased invasion by G‐Gly. Together, these results demonstrate that G‐Gly renders colon cancer cells more invasive by increasing MMP‐1 and MMP‐3 expressions via the putative G‐Gly receptor and would thus be a good molecular target in a clinical setting. © 2004 Wiley‐Liss, Inc.
mTHPC‐mediated photodynamic therapy for early oral squamous cell carcinoma
Authors: Hopper Colin , Kübler Alexander , Lewis Harry , Tan I. Bing , Putnam Graham ,   the Foscan 01 Study Group
Published: June 2004

Surgery and radiotherapy are standard treatments for early oral squamous cell carcinoma, both resulting in good tumour control. However, neither of these modalities is without consequent functional or cosmetic impairment, and there are patients in whom both are contraindicated. Furthermore, there is a significant risk of metachronous tumours developing in the oral cavity, and salvage or retreatment with either surgery or radiotherapy poses difficulties. Photodynamic therapy (PDT) offers the potential for improved functional and cosmetic outcomes, while achieving comparable tumour control. We conducted an open‐label, multicentre study to assess the efficacy and safety of meta‐tetrahydroxyphenylchlorin (mTHPC) in patients with early oral cancer. One hundred twenty‐one patients received intravenously administered mTHPC, followed 96 hr later by illumination of the tumour surface with 652 nm laser light. Of these patients, 114 were protocol compliant. A complete tumour response was achieved in 85% of protocol‐compliant patients (97 of 114 patients). A complete response was maintained in 85% of responders at 1 year and in 77% at 2 years. One‐ and 2‐year actuarial survival rates were 89% and 75%, respectively. In the opinion of the investigators, tumour clearance was accompanied by excellent cosmetic and functional results, without impact on the patients' performance status. Mild‐to‐moderate pain at the treatment site, a recognised side effect of PDT in the oral cavity, was reported by 82% of patients but was manageable with appropriate analgesia. Mild‐to‐moderate skin photosensitivity reactions were reported for 13% of patients. mTHPC offers an effective alternative treatment for early oral squamous cell carcinoma. It is associated with excellent functional and cosmetic results and can be used in conjunction with other standard therapies. © 2004 Wiley‐Liss, Inc.
C8orf4 is a transforming growth factor B induced transcript downregulated in metastatic colon cancer
Authors: Friedman Joshua B. , Brunschwig Elaine B. , Platzer Petra , Wilson Keith , Markowitz Sanford D.
Published: June 2004

Transforming growth factor (TGF) β mediates a tumor suppressor pathway in human colon epithelial cells. We were interested in identifying and characterizing novel genes regulated by the TGFβ pathway in the colon. We employed expression microarrays to identify transcripts induced by TGFβ in Vaco 330, a colon adenoma cell line. We then used expression microarrays to determine which of these TGFβ induced transcripts are down‐regulated in metastatic colon cancer. Northern analysis and real‐time reverse transcription PCR confirmed and quantified our findings from the microarrays. These analyses highlighted C8orf4 as induced by TGFβ in colon cells. Moreover, C8orf4 is expressed in most normal colon mucosa samples, and is not expressed in most colon cancer metastases or colon cancer cell lines. Colon cancer primary tumors showed reduced expression of C8orf4 relative to normal mucosa, possibly reflecting contributions of C8orf4 expression in stromal cells. C8orf4 is a gene regulated by TGFβ signaling and loss in advanced colon cancer suggests C8orf4 may play a role in colon cell differentiation or growth regulation. © 2004 Wiley‐Liss, Inc.
Altered mode of allelic replication accompanied by aneuploidy in peripheral blood lymphocytes of prostate cancer patients
Authors: Dotan Zohar A. , Dotan Aviva , Ramon Jacob , Avivi Lydia
Published: June 2004

Replication timing of the genetic material is a highly programmed process correlated with expression, stability and methylation capacity. An important aspect of that timing is the temporal order of allelic replication: a synchronous mode for biallelically expressed genes and an asynchronous for monoallelically expressed genes. Previous studies showed that malignancy is associated with changes in the inherent mode of allelic replication, and even normal cells of cancer patients display alterations in the replication of various genes. Using fluorescence in situ hybridization (FISH), we checked whether allelic‐replication mode differentiates cancer patients from healthy individuals. We focused on prostate cancer (CAP), the most common diagnosed cancer and the second leading cause of cancer death in men over 50 years old. Five nonrelated genes and a nontranscribed DNA sequence associated with chromosomal segregation were used in our study. All 6 tested loci displayed in peripheral blood lymphocytes stimulated with phytohemagglutinin (PHA) of CAP patients loss of their inherent temporal order of allelic replication, coupled with aneuploidy, the outcome of chromosome malsegregation. The replication‐timing modification is a reversible epigenetic alteration, evidenced by our ability to resurrect the normal pattern in all 6 tested loci by introducing an inhibitor of methyl transferase. On the other hand, the methylation‐blocking agent failed to obliterate aneuploidy. The replication alteration accompanied by aneuploidy, detected in peripheral blood cells, distinguishes between CAP patients and individuals with benign prostate hyperplasia (BPH; a common disorder in elderly men) better than the routinely used blood marker, the prostate‐specific antigen (PSA). © 2004 Wiley‐Liss, Inc.
Microsatellite analysis of serum DNA in patients with head and neck cancer
Authors: Nawroz‐Danish Homaira , Eisenberger Claus F. , Yoo George H. , Wu Li , Koch Wayne , Black Carri , Ensley John F. , Wei Wei‐Zen , Sidransky David
Published: June 2004

We have shown previously that microsatellite alterations in serum DNA was predictive of distant metastasis in a study with 21 primary head and neck squamous cell carcinoma patients. To further investigate serum microsatellite alterations as a prognostic tool, we carried out microsatellite analysis of serum DNA with 10 markers on 152 patients with head and neck cancer. Forty‐five percent (68/152) of patients had microsatellite alterations of serum DNA identical to corresponding tumor DNA. In 16 patients that had distant metastasis, 11 patients had a positive serum test (microsatellite alterations detectable in their serum DNA with one or more markers). The difference in distant metastasis rates between the negative and positive serum tests (6.0% [5/84] vs. 16.2% [11/68], RR = 2.7) was clinically significant and almost reached statistical significance (p = 0.06). When the analysis was restricted to patients with recurrent disease, a positive serum test correlated with those who developed distant metastasis (p = 0.04). Other parameters, such as development of recurrence, stage of the cancer, disease‐free survival and overall survival, were not associated with a positive serum test. Detecting tumor DNA in serum by microsatellite analysis may help identify patients at risk for distant metastasis. Therefore, circulating tumor cells may contribute to the presence of tumor DNA in the serum. In the future if a serum test is positive, therapeutic approaches may by intensified, such as platinum‐based chemoradiation, to reduce distant failures. © 2004 Wiley‐Liss, Inc.
Determination of the rat tissue partitioning of endotoxin in vitro for physiologically‐based pharmacokinetic (PBPK) modeling
Authors: Ross Ivan A. , Sapienza Philip P. , Hanes Darcy E. , Johnson Widmark , Kim Chung S.
Published: June 2004

The biosynthetically double‐labeled lipopolysaccharide (LPS), containing 3H‐labeled on the fatty acyl‐chains and 14C‐labeled on the glucosamine of Salmonella enterica serotype typhimurium, was isolated from bacteria grown in proteose peptone‐beef extract (PPBE) medium in the presence of labeled precursors; 133 µCi/ml of [2‐3H] acetate sodium salt and 0.167 µCi/ml of N‐acetyl[D‐1‐14C]glucosamine. The LPS was extracted from the bacteria with 90% phenol/chloroform/petroleum ether, puriﬁed and stored in 0.1% (v/v) triethylamine/10 mM Tris HCl at −70 °C. Tissue slices and portions of the meninges were prepared and incubated in artiﬁcial cerebrospinal ﬂuid (CSF) or Krebs phosphate buffer (Krebs) containing 150 ng/ml LPS with [3H] LPS (0.004 µCi/ml, sp. act. 28 µCi/mg LPS). The tissues were incubated under 95% oxygen/5% carbon dioxide at 37 °C with constant agitation until steady‐state uptake was reached (60 min). At the end of the incubation period, tissues were processed for radioactivity measurement. The rat tissue partitioning of LPS in artiﬁcial CSF for brain and Krebs for other organs was measured by using the ratio of tissue to medium at the steady state in vitro. The following results were obtained from the study: Heart, 0.15; liver, 0.19; spleen, 0.12; kidney, 0.18; stomach, 0.17; small intestine, 0.18; brain stem, 0.10; cerebellum, 0.11; meninges, 0.77; hippocampus, 0.12; hypothalamus, 0.12; frontal cortex, 0.09 and caudate nucleus, 0.10. This information, along with plasma or blood/buffer partition coefﬁcients, is a requisite for constructing a physiologically‐based pharmacokinetic (PBPK) model of endotoxins for quantitative risk assessment. Copyright © 2004 John Wiley & Sons, Ltd.
Inhibition of topoisomerase II in 32D.3(G) cells by hydroquinone is associated with cell death
Authors: Fung Jacqueline , Hoffmann Matthew J. , Kim David D. , Snyder Robert
Published: June 2004

Studies showing the inhibition of isolated human topoisomerase II (topo II) by benzene metabolites such as hydroquinone, coupled with the recognition that benzene‐induced acute myelogenous leukemia bears a resemblance to second cancers caused by topo II inhibitors such as etoposide, suggested that topo II inhibition by hydroquinone might induce leukemogenic mutations. In these studies the inhibition of topo II by hydroquinone or etoposide was studied in parallel with the effects of these agents on differentiation, maturation and viability in murine bone marrow 32D.3(G) cells. Topoisomerase II of 32D.3(G) cells was inhibited by hydroquinone at concentrations of 5 µM or higher and by etoposide at concentrations of 50 µM or higher. At concentrations of either agent below those that inhibited topo II the cells responded normally to interleukin‐3, which promoted proliferation, and to granulocyte colony‐stimulating factor, which promoted differentiation and maturation. In dose ranges in which topo II was inhibited by either hydroquinone or etoposide, the cells became progressively less viable and cell counts decreased during the incubation period. Progressive inability to detect topo II protein by Western blot analysis as hydroquinone concentrations were increased suggested that either association of the probe with the enzyme was inhibited by hydroquinone or there was degradation of the protein as a function of hydroquinone‐induced apoptosis. Copyright © 2004 John Wiley & Sons, Ltd.
Reactions of nifurtimox with critical sulfhydryl‐containing biomolecules: their potential toxicological relevance
Authors: Díaz E. G. , Montalto de Mecca M. , Castro J. A.
Published: June 2004

Nifurtimox (Nfx) is a drug used in the treatment of Chagas' disease, an endemic parasitic disease from Latin American countries. It produces undesirable side‐effects in patients, frequently forcing the treatment to be stopped. Its toxic mechanism is not fully understood. In this work we describe purely chemical reactions of Nfx with relevant cellular sulfhydryl (SH) compounds. The compounds tested were glutathione (GSH), cysteine (RSH), lipoic acid (LA) and coenzyme A (CoA). All reacted with Nfx to give nitrite (NO−2). The relative reaction rates were CoA>LA>GSH>RSH. In studies with GSH and RSH the formation of nitrite was accompanied by decreases in Nfx concentration and increases in the formation of a reaction product revealed by HPLC. We failed to show the presence of liver cytosolic GST (GSH transferase activity)‐mediated formation of NO2‐ from Nfx. These NO−2‐releasing processes occurred under in vivo conditions in Nfx‐treated Sprague‐Dawley male rats (240–260 g body weight) at a dose of 100 mg Nfx kg−1 p.o. In urine samples NO−2 excretion was accompanied by unchanged drug and two unidentiﬁed more polar metabolites detectable by HPLC. The Nfx reactions with critical SH from molecules such as GSH, RSH, LA and CoA, and potentially others containing SH residues (e.g. enzymes or structural proteins), might have toxicological relevance not only for the Nfx side‐effects but also for the chemotherapeutic effects on Trypanosoma cruzi. In addition, Nfx reactions with GSH might be crucial in Nfx detoxiﬁcation. Copyright © 2004 John Wiley & Sons, Ltd.
Ultrastructural changes induced in HeLa cells after phototoxic treatment with harmine
Authors: Pérez Martín J. M. , Labrador V. , Fernández Freire P. , Molero M. L. , Hazen M. J.
Published: June 2004

In the present work, we have continued our studies on harmine phototoxicity in human tumour cells. The toxicity of harmine in the dark was analysed by a quantitative neutral red uptake assay, and subcellular sensitive targets following harmine photosensitization were deﬁned by electron microscopic analysis of HeLa cells. The results obtained indicated that this compound shows a clear dose‐dependent cytotoxic effect in the dark. The combined treatment with suitable doses of harmine and UV radiation was very effective at an early stage, although maximal cell killing appeared 48 h after photodynamic activation. Ultrastructural examination of HeLa cells immediately after the photodynamic treatment revealed lysosomal destabilization and profound cytoplasmic vacuolization that evolved to cytolysis, which is typical of necrotic cell death. It is concluded that harmine could be a valuable photosensitizer whose biological applications merit further evaluation. Copyright © 2004 John Wiley & Sons, Ltd.
Bioactivation, toxicokinetics and acute effects of chloroform in fisher 344 and Osborne Mendel Male rats
Authors: Gemma Simonetta , Testai Emanuela , Chieco Pasquale , Vittozzi Luciano
Published: June 2004

Chloroform has been regarded as a renal carcinogen, based on results obtained with Osborne Mendel (OM) rats. Fisher 344 (F344) rats, considered representative of OM rats on the basis of comparable acute toxic effects, have been used in most of the studies aimed to elucidate the mechanisms of kidney tumour induction. In the present work, in vitro and in vivo chloroform bioactivation in the liver and kidney of F344 and OM rats has been reported, as well as additional toxicokinetics and acute toxicity information. Complete similarity of chloroform metabolism and toxicokinetics was evidenced in the two rat strains. Chloroform metabolism was fully saturated at the OM rat bioassay doses (90–180 mg kg−1 body wt.), working at a maximal rate of 40–50 µmol 14CO2 expired kg−1 h−1. No acute hepatotoxicity, nephrotoxicity or consequent cell proliferation was evidenced at 180 mg kg−1 body wt. chloroform. In the rat liver, phosgene was conﬁrmed as the major metabolite. Renal microsomes from both F344 and OM rats in vitro were unable to produce any oxidative metabolite; at variance, adducts due to oxidative and reductive metabolites were detected in vivo. Our results indicated the presence in the rat kidney of electrophilic metabolites other than phosgene, representing either oxidative metabolites formed elsewhere and sufﬁciently stable to be transported to the kidney or electrophilic metabolites secondary to the formation of reductive radicals. Therefore, the rat kidney represents a suitable model to study the toxicological effects, including genotoxicity, of chloroform metabolites in the absence of cytotoxic effects produced by phosgene formed in situ. Copyright © 2004 John Wiley & Sons, Ltd.
Effects of ochratoxin a on oxidative damage in rat kidney: protective role of melatonin
Authors: Özçelik Nurten , Soyöz Mustafa , Kılınç İbrahim
Published: June 2004

Epidemiological studies indicate that ochratoxin A (OTA) may be involved in the pathogenesis of different forms of human nephropathies. Melatonin (MEL) displays antioxidant and free radical scavenger properties. In the present study, the effect of MEL on the oxidative stress induced by OTA administration in rats was investigated. Three groups of eight rats each were used: control, OTA (289 µg kg−1 day−1) and OTA + MEL (OTA, 289 µg kg−1 day−1; MEL, 10 mg kg−1 day−1), with treatment at two different time periods during the same day. After 4 weeks of treatment, the levels of lipid peroxidation (LPO) and the activities of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) were measured in homogenates of kidney. In OTA‐treated rats, the levels of LPO and the activities of GSHPx and SOD were signiﬁcantly decreased compared with controls. In OTA + MEL‐treated rats, SOD, GSHPx and catalase activities and LPO levels were not changed signiﬁcantly in comparison with controls. Copyright © 2004 John Wiley & Sons, Ltd.
QT prolongation in anaesthetized guinea‐pigs: an experimental approach for preliminary screening of torsadogenicity of drugs and drug candidates
Authors: Testai L. , Calderone V. , Salvadori A. , Breschi M. C. , Nieri P. , Martinotti E.
Published: June 2004

Many non‐cardiovascular drugs can prolong the QT interval of the electrocardiogram (ECG); this is an accessory property not necessary for their pharmacological action and generally linked to the block of the potassium HERG channels and delayed cardiac repolarization. The QT prolongation can lead to a dangerous tachyarrhythmia, called torsade de pointes, and potentially to fatal ventricular ﬁbrillation. The experimental approaches, aimed at an early identiﬁcation of this undesidered property, often require sophisticated and expensive equipment or the use of superior animal species (dog, primates) that cannot be employed easily for ethical and/or economic reasons.

This work aimed to study drug‐induced QT prolongation in anaesthetized guinea‐pigs and to evaluate the reliability of such an experimental approach to obtain a satisfying predictive parameter of the torsadogenicity of drugs in humans. Seven drugs that were torsadogenic in humans (astemizole, cisapride, haloperidol, quinidine, sotalol, terfenadine and thioridazine) and two that were non‐torsadogenic (chlorprotixene and diazepam) were administered i.v. to guinea‐pigs under pentobarbital anaesthesia. The ECGs were recorded by four electrodes inserted in the subcutaneous layer of the limbs. Both RR and QT intervals were measured in Leads II and III and then the correct QT values were calculated by Bazett and Fridericia algorithms (QTcB and QTcF, respectively). All the drugs, with the exception of chlorprotixene and diazepam, produced a dose‐dependent prolongation of the QT and RR intervals and a signiﬁcant increase of QTcB and QTcF values. It can be concluded that this method represents a rapid and low‐cost procedure to evaluate the cardiac safety proﬁle in the preliminary screening of a high number of drugs or drug candidates. Copyright © 2004 John Wiley & Sons, Ltd.
Evaluation of lipid peroxidation in red blood cells by monitoring the uptake of sucrose and phenol red
Authors: Sawas A. H. , Pentyala S. N.
Published: June 2004

Red blood cells (RBCs) are prone to lipid peroxidation by virtue of their function as oxygen carriers, and also because of their lipid composition. Malondialdehyde (MDA) content using thiobarbituric reagent is widely used to quantify lipid peroxidation. In this study we compare MDA assay with a newly developed assay that evaluates the uptake of sucrose and phenol red into RBCs under peroxidative stress. Both sucrose and phenol red uptake show signiﬁcantly higher correlation with incubation time compared with MDA assay. Furthermore, phenol red uptake into RBCs on treatment with H2O2 has a direct linear proportional relationship, whereas it is hyperbolic with MDA. The assay also clearly shows that uptake of sucrose or phenol red is speciﬁc for intact cells (RBCs) prior to hemolysis. Assay validation is carried out by using known lipid peroxidation‐causing agents, such as ferrous ions, and also by using peroxidation inhibitors such as α‐tocopherol. This new method can be applied efﬁciently to evaluate lipid peroxidation in RBCs as well as other cells and tissues. Copyright © 2004 John Wiley & Sons, Ltd.
Pulmonary irritant potency of polyisocyanate aerosols in rats: comparative assessment of irritant threshold concentrations by bronchoalveolar lavage
Authors: Pauluhn Jürgen
Published: June 2004

The object of this study was to compare the relative acute pulmonary irritant potency of respirable aerosols of a variety of non‐volatile polyisocyanates. The types of polyisocyanates examined included aliphatic homopolymers and mixed aliphatic–aromatic polyisocyanates consisting of the following monomers: HDI (hexamethylene 1,6‐diisocyanate), IPDI (isophorone diisocyanate), MDI (methylene‐diphenyl‐4,4′‐diisocyanate) and TDI (toluene diisocyanate). For reference purposes, the pulmonary irritant polyisocyanate aerosols were compared with monomeric IPDI, a semi‐volatile respiratory tract (airway) irritant. In the substances tested, the concentration of free isocyanate moieties ranged from 11% to 38%. The relative potency to elicit pulmonary irritation was assessed by measurements of lung weights and total protein and lactate dehydrogenase (LDH) in the bronchoalveolar lavage ﬂuid (BALF) following a single 6‐h exposure of male rats. The time course of changes was analysed 3 h and 1, 3 and 7 days after exposure. When exposed to irritant concentrations of aerosol, BALF protein was maximal on post‐exposure day 1 and returned to the level of the controls on post‐exposure day 7. In contrast, rats exposed to sub‐lethal concentrations of monomeric IPDI experienced a time‐related increase in BALF protein. Based on this most sensitive endpoint, extrapolated no‐observed‐effect concentrations (NOECs) were in the range of 2–3 mg m−3 for most polyisocyanates examined. The NOECs from all the substances investigated were in the range 1–50 mg m−3. Thus, this methodology is adequate to rank the pulmonary irritant potency of polyisocyanate aerosols and to differentiate pulmonary from airway irritants. For pulmonary irritants the estimated acute NOECs were essentially similar to the no‐observed‐adverse effect concentrations (NOAECs) from long‐term, repeated‐exposure inhalation studies available for some of the polyisocyanates. A clear dependence of the NOAECs on the content of free isocyanate moieties was not observed. In summary, it is concluded that pulmonary irritation caused by polyisocyanate aerosols can be quantiﬁed readily in an acute rat bioassay by analysis of total protein in BALF. Moreover, this experimental evidence suggests that the NOECs of pulmonary irritants based on this endpoint are predictive of the NOAECs observed after subacute/subchronic inhalation exposure, suggesting that acute pulmonary irritation governs the outcome of repeated inhalation studies with such aerosols. However, for isocyanates where airway irritation predominates the pulmonary irritation, long(er)‐term inhalation studies appear to be indispensable. The content of free NCO per se appears to be a poor predictor of the pulmonary irritant potency of polyisocyanate aerosols. Copyright © 2004 John Wiley & Sons, Ltd.