bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence and physical status of human papillomavirus in squamous cell carcinomas of the head and neck
Authors: Koskinen Walter J. , Chen Ren Wei , Leivo Ilmo , Mäkitie Antti , Bäck Leif , Kontio Risto , Suuronen Riitta , Lindqvist Christian , Auvinen Eeva , Molijn Anco , Quint Wim G. , Vaheri Antti , Aaltonen Leena‐Maija
Published: September 2003

Fresh‐frozen biopsies were obtained from 61 patients at diagnosis of squamous cell carcinoma of the head and neck (HNSCC) for study of the prevalence and physical status of human papillomavirus (HPV) DNA. The frequency of HPV DNA and genotypes were determined by SPF10 PCR screening with a general probe hybridization and INNO‐LiPA HPV genotyping assay. In addition, a single‐phase PCR with primers FAP 59/64 and a nested PCR with primers CP 65/70 and CP 66/69 served to detect particularly cutaneous HPV types. By the sensitive SPF10 PCR and INNO‐LiPA assay, 37 of 61 (61%) samples were positive for HPV. HPV‐16 was the most frequently detected type (31 of 37, 84%). Multiple infections were found in 8 of 37 (22%) of the HPV‐positive samples, and co‐infection by HPV‐16 and HPV‐33 was predominant. No cutaneous HPV types were detected. Patients with HPV‐positive tumors had similar prognosis as those with HPV‐negative ones. Real‐time PCR analysis of the HPV‐16 positive samples indicated the presence of integrated (11 of 23, 48%), episomal (8 of 23, 35%) and mixed forms (4 of 23, 17%) of HPV DNA. The viral load of HPV DNA exhibited large variation. The median copy numbers of E6 DNA in tonsillar specimens were approximately 80,000 times higher than that in nontonsillar HNSCC ones. Patients with episomal viral DNA were more frequently found to have large (T3–T4) tumors at diagnosis than were those with integrated or mixed forms. © 2003 Wiley‐Liss, Inc.
A subset of head and neck squamous cell carcinomas exhibits integration of HPV 16/18 DNA and overexpression of p16INK4A and p53 in the absence of mutations in p53 exons 5–8
Authors: Hafkamp Harriet C. , Speel Ernst J.M. , Haesevoets Annick , Bot Fredrik J. , Dinjens Winand N.M. , Ramaekers Frans C.S. , Hopman Anton H.N. , Manni Johannes J.
Published: September 2003

Besides well‐known risk factors such as tobacco use and alcohol consumption, oncogenic human papillomavirus (HPV) infection also has recently been suggested to promote head and neck tumorigenesis. HPV is known to cause cancer by inactivation of cell cycle regulators p53 and pRb via expression of viral oncoproteins E6 and E7. This indicates that p53 mutations are not a prerequisite in HPV‐induced tumor development. However, discrepancy exists with respect to the frequency of head and neck squamous cell carcinomas (HNSCC) harboring DNA of oncogenic HPV and the fraction of these tumors showing p53 mutations. In our study, we examined the frequency of HNSCC demonstrating HPV 16/18 integration as identified by fluorescence in situ hybridization (FISH) and investigated their p53 (mutation) status by immunohistochemistry and single‐strand conformation polymorphism (SSCP) analysis of exons 5–8. Paraffin‐embedded, archival biopsy material from 27 premalignant mucosal lesions and 47 cases of HNSCC were analyzed. Ten of the 47 (21%) HNSCC unequivocally exhibited HPV 16 integration, including 8 of 12 (67%) tonsillar carcinomas. This is supported by the immunohistochemical detection of p16INK4A overexpression in all 10 HPV‐positive tumors. Although FISH is considered to be less sensitive than PCR‐based methods for HPV detection, our data clearly demonstrate clonal association of HPV with these tumors, as illustrated by the presence of integrated HPV 16 in both the primary tumor and their metastases in 2 patients. In contrast, HPV 16/18 DNA could not be detected in the premalignant lesions. In 30 of 47 (64%), HNSCC accumulation of p53 was observed, including 8 of the 10 HPV‐positive carcinomas. However, in none of the latter cases could mutations in exons 5–8 be identified, except for a polymorphism in codon 213 of exon 6 in one patient. Evaluation of clinical data revealed a significant inverse relation between tobacco use with or without alcohol consumption, and HPV positivity of the tumors. © 2003 Wiley‐Liss, Inc.
Aberrant methylation of the cyclin D2 promoter in primary small cell, nonsmall cell lung and breast cancers
Authors: Virmani Arvind , Rathi Asha , Heda Shashank , Sugio Kenji , Lewis Cheryl , Tonk Vijay , Takahashi Takashi , Roth Jack A. , Minna John D. , Euhus David M. , Gazdar Adi F.
Published: September 2003

DNA methylation alteration of several genes contributes to human tumorigenesis. Cyclin D2, a member of the D‐type cyclins, is implicated in cell cycle regulation and malignant transformation. In our study, we examined the methylation status of the cyclin D2 promoter in small cell lung cancer (SCLC), nonsmall cell lung cancer (NSCLC), breast tumors and tumor cell lines. We observed that aberrant methylation of cyclin D2 was present in 32 of 56 (57%) SCLC cell lines, 7 of 32 (22%) SCLC tumor tissues; 25 of 61 (47%) NSCLC cell lines, 19 of 48 (40%) NSCLC tumor tissues; 18 of 30 (60%) breast tumor cell lines and 19 of 63 (30%) breast tumor tissues. Methylation was more frequent in the tumor cell lines compared to the primary breast and SCLC tumors (p = 0.007 and p = 0.001, respectively). Methylation was rare in the control tissue samples; 0 of 12 peripheral blood lymphocytes; 0 of 12 buccal epithelial cells; 0 of 18 nonmalignant lung tissues and 3 of 28 (11%) nonmalignant breast tissues. Promoter methylation correlated with loss of transcript by reverse transcription PCR (RT‐PCR) in 9 of 11 (6 lung, 5 breast) tumor cell lines tested. Two cell lines that were not methylated also lacked expression, suggesting that other mechanisms of inactivation may be involved. Expression was restored by treatment with the demethylating agent, 5 aza 2′ deoxycytidine, in all 9 methylated cell lines. Our results confirm earlier reports in breast cancer and indicate that aberrant methylation of cyclin D2 may contribute to the pathogenesis of the 2 major types of lung cancers. © 2003 Wiley‐Liss, Inc.
Whole bladder wall photodynamic therapy of transitional cell carcinoma rat bladder tumors using intravesically administered hypericin
Authors: Kamuhabwa Appolinary A.R. , Roskams Tania , D'Hallewin Marie‐Ange , Baert Luc , Van Poppel Hein , de Witte Peter A.M.
Published: September 2003

Whole‐bladder wall photodynamic therapy (PDT) is a promising treatment for carcinoma in situ (CIS) and diffuse premalignant changes of the bladder. After the results of our clinical studies showing that intravesical hypericin selectively accumulates in superficial bladder tumors, we investigated the hypericin‐PDT efficacy in an AY‐27 orthotopic transitional cell carcinoma rat bladder tumor model. After the instillation of hypericin (30 μM, 2 hr) in the bladder, tumors were irradiated (25–50 mW/cm 6–48 J/cm2) using 595 nm laser light. Data demonstrate that light doses of 12–48 J/cm2 resulted in selective PDT‐induced urothelial tumor damage without damaging detrusor musculature. Histological assessment of bladder sections 2 days after PDT showed tumor destruction, with tumor cells shrinking and detaching from the bladder wall. There were tumor regrowth 1–3 weeks after treatment. The in vivo/in vitro clonogenic assay results revealed up to 98% of tumor cell kill by hypericin PDT. In conclusion, hypericin PDT can be used to safely induce a selective urothelial tumor damage without damaging detrusor musculature, when optimum hypericin concentration and light fluences are used. A small percentage (2–5%) of tumor cells that survive the photodynamic treatment resulting in tumor regrowth after a prolonged period of time is likely due to oxygen depletion during light irradiation. © 2003 Wiley‐Liss, Inc.
Site‐specific human breast cancer (MDA‐MB‐231) metastases in nude rats: Model characterisation and in vivo effects of ibandronate on tumour growth
Authors: Neudert Marcus , Fischer Christian , Krempien Burkhard , Bauss Frieder , Seibel Markus J.
Published: September 2003

Animal models are important tools to study the development of bone metastases and to evaluate strategies for their prevention and treatment. We here describe a new model in which tumour inoculation is achieved by injection of cancer cells into the femoral artery. This approach results in the development of multiple osteolytic lesions in the distal femora and proximal tibiae within 18 days after inoculation, with a success rate of 95–100% and no additional comorbidity. In untreated animals, osteolyses expanded continuously at a growth rate of 4.7–8.2 mm2/4 days, causing extensive destruction of resident bone structures by the tumour, significant loss of tibial bone density and a transient rise in urinary bone resorption markers. Continuous daily treatment with ibandronate (10 μg/kg) inhibited further growth of fully established metastases and reduced the mean osteolytic growth rate to 0.03 mm2/4 days. In lesions <6 mm bisphosphonate treatment resulted in a negative growth rate (−0.33 to −0.81 mm2/4 days). When ibandronate was started 3 days prior to tumour cell inoculation, the development of osteolytic lesions was substantially reduced (take rate only 17%) and bone density and structure were mostly preserved. We conclude that the intra‐arterial approach used in this new model of metastatic bone disease results in site‐specific osteolytic lesions with high take rates, steady tumour growth and no additional morbidity. While serial bone marker assessments did not prove useful to monitor osteolytic growth, our studies provide in vivo evidence that ibandronate treatment induces tumour remission by reversal of tumour growth. © 2003 Wiley‐Liss, Inc.
Overexpression of histone deacetylase HDAC1 modulates breast cancer progression by negative regulation of estrogen receptor α
Authors: Kawai Hideki , Li Huchun , Avraham Shalom , Jiang Shuxian , Avraham Hava Karsenty
Published: September 2003

The interaction between 17β‐estradiol and estrogen receptor alpha (ER‐α) plays an important role in breast carcinogenesis and breast cancer treatment. ER‐α is a critical growth regulatory gene in breast cancer and its expression level is tightly linked to the prognosis and treatment outcomes of breast cancer patients. Loss of ER‐α expression in breast epithelial cells is critical for breast cancer progression. The underlying molecular mechanisms for this loss, however, are poorly defined. Histone deacetylases (HDACs) are implicated in the alteration of chromatin assembly and tumorigenesis. We show that histone deacetylase 1 (HDAC1) interacts with ER‐α in vitro and in vivo and suppresses ER‐α transcription activity. The interaction of HDAC1 with ER‐α was mediated by the AF‐2 and DBD domains of ER‐α. We observed an endogenous interaction of HDAC1 with ER‐α in breast cancer cells, which was decreased in the presence of estrogen. Interestingly, overexpression of HDAC1 in stable transfected MCF‐7 clones induced loss of ER‐α and significantly increased cell proliferation and colony formation, as compared to the control MCF‐7 cells, whereas treatment of stable MCF‐7 clones with the HDAC specific inhibitor trichostatin A (TSA) induced re‐expression of ER‐α mRNA and protein. Our findings strongly suggest that HDAC1 affects breast cancer progression by promoting cellular proliferation in association with a reduction in both ER‐α protein expression and transcriptional activity. Thus, HDAC1 may be a potential target for therapeutic intervention in the treatment of a subset of ER‐negative breast cancers. © 2003 Wiley‐Liss, Inc.
Centrosomal aberrations in primary invasive breast cancer are associated with nodal status and hormone receptor expression
Authors: Schneeweiss Andreas , Sinn Hans‐Peter , Ehemann Volker , Khbeis Tanja , Neben Kai , Krause Ulf , Ho Anthony D. , Bastert Gunther , Krämer Alwin
Published: September 2003

Our purpose was to assess the presence of centrosomal aberrations as measured by immunohistochemistry in primary invasive breast cancer and their association with established and proposed prognostic factors. Tissue sections of 103 primary invasive breast cancers were examined using centrosome‐specific antibodies to pericentrin and γ‐tubulin. At least 3 different tumor regions per case were examined to determine maximum centrosomal aberration levels, which represent the proportion of cells with abnormal centrosomes in the region with the highest percentage of cells with centrosomal aberrations. The χ2 test was performed to evaluate the association of maximum centrosomal aberration levels with patient age; tumor size; nodal status; nuclear grade; hormone receptor and Her2/neu expression; proportion of Ki67‐, p53‐ and Bcl‐2‐positive tumor cells; DNA index; S‐phase fraction; and proliferation index. With pericentrin immunohistochemistry, maximum centrosomal aberration levels >35% were detectable in 92 of the 103 breast carcinomas (89%). We found a highly significant correlation of maximum centrosomal aberration levels above 35% with axillary nodal tumor involvement (p < 0.0001) and the absence of hormone receptors (p < 0.0001). In addition, there was a borderline significant relationship with age <50 years (p = 0.050) and Her2/neu overexpression (p = 0.050). Among node‐negative patients, maximum centrosomal aberration levels >35% were also associated with an increased DNA index (p = 0.006). In a subset of patients, additional staining of centrosomes with a monoclonal anti‐γ‐tubulin antibody essentially confirmed these results. In primary invasive breast cancer, centrosomal aberrations are associated with those factors predicting a more aggressive course of disease. This might indicate a fundamental role of centrosomal dysfunction in disease evolution, possibly as a result of chromosome missegregation during mitosis. © 2003 Wiley‐Liss, Inc.
The synthetic retinoid adapalene inhibits proliferation and induces apoptosis in colorectal cancer cells in vitro
Authors: Ocker Matthias , Herold Christoph , Ganslmayer Marion , Hahn Eckhart G. , Schuppan Detlef
Published: September 2003

Chemotherapy of advanced stages of colorectal carcinoma is unsatisfactory. Retinoids inhibit cell growth and induce apoptosis in a variety of human malignancies. We compared the effect of the synthetic retinoid adapalene (ADA) and 9‐cis‐retinoic acid (CRA) on carcinoma cell lines in vitro. Colon carcinoma cell lines CC‐531, HT‐29 and LOVO as well as human foreskin fibroblasts were exposed to different concentrations of ADA and CRA for 3–72 hr. Proliferation was assessed by BrdU incorporation and apoptosis by FACS analysis. Breakdown of ΔΨm was determined by JC‐1 staining and activity of caspases 3 and 8, by a colorimetric assay. Quantitative Western blots were performed to detect changes in bax, bcl‐2 and caspase‐3. Both retinoic derivatives suppressed DNA synthesis and induced apoptosis in all tested cell lines time‐ and dose‐dependently. While the natural retinoid CRA showed moderate antiproliferative and proapoptotic effects only at the highest concentration (10–4 M), the synthetic retinoic ADA was significantly more effective, showing remarkable effects even at 10–5 M. ADA and CRA disrupt ΔΨm and induce caspase‐3 activity in responsive tumor cells. Quantitative Western blots showed a shift of the bax:bcl‐2 ratio toward proapoptotic bax in ADA‐treated cells. Our results clearly indicate the superiority of ADA compared to CRA. Therefore, we suggest that ADA may be far more suitable as an adjunctive therapeutic agent for treatment of colon cancer in vivo. © 2003 Wiley‐Liss, Inc.
Selective suppression of monocyte chemoattractant protein‐1 expression by human papillomavirus E6 and E7 oncoproteins in human cervical epithelial and epidermal cells
Authors: Kleine‐Lowinski Kerstin , Rheinwald James G. , Fichorova Raina N. , Anderson Deborah J. , Basile John , Münger Karl , Daly Christine M. , Rösl Frank , Rollins Barrett J.
Published: September 2003

Infection of cervical keratinocytes by high‐risk HPV is involved in the etiology of cervical carcinoma. Since viral products are immunogenic, development of cancer may require suppression of immune responses directed against infected epithelial cells. Many markers of host immune effector responses decrease as cervical intraepithelial neoplasia progresses. Among these is epithelial cell expression of the chemokine MCP‐1, though the mechanism for its suppression is unclear. Here, we show that the E6 and E7 viral oncogenes from high‐risk HPV, individually and together, suppress MCP‐1 expression in primary epithelial cells derived from the female genital tract. This is not a consequence of global suppression of chemokine expression since other chemokines, including IP‐10, IL‐8 and RANTES, were less affected. Furthermore, 4 of 6 HPV‐positive cervical carcinoma cell lines did not express MCP‐1. Our data indicate that suppression of MCP‐1 expression is part of the program of high‐risk HPV E6/E7‐induced transformation of primary epithelial cells. These observations are consistent with a model in which MCP‐1 expression by infected keratinocytes, which would stimulate an immune attack on HPV‐transformed cells, is suppressed for invasive cervical cancer to appear. © 2003 Wiley‐Liss, Inc.
Air pollution and lung cancer mortality in the vicinity of a nonferrous metal smelter in Sweden
Authors: Bessö Anna , Nyberg Fredrik , Pershagen Göran
Published: September 2003

To evaluate the importance of exposure to ambient air pollution for lung cancer risk, we conducted a case‐control study in the vicinity of a nonferrous metal smelter. The smelter started operations in 1930 and had very high emissions during the early decades, particularly of arsenic and SO2. Among subjects deceased 1961–1990 in the municipality where the smelter is located and who had not worked at the smelter, 209 male and 107 female lung cancer cases were identified and matched by sex and year of birth to 518 and 209 controls, respectively. Information on smoking habits, occupations and residences was collected by questionnaire to next‐of‐kin and from registry data. Living close to the smelter was associated with a relative risk (RR) for lung cancer of 1.38 [95% confidence interval (CI) 0.89–2.14] among men, adjusted for smoking and occupational exposures. No clear difference in risk was detected for men deceased 1961–1979 compared to men deceased 1980–1990 (RR point estimates 1.42 and 1.29, respectively). There appeared to be an increased risk especially for men exposed in the beginning of the operations (RR = 1.51, 95% CI 0.90–2.54), in particular combined with exposure duration shorter than 20 years (RR = 2.52, 95% CI 0.89–7.11). For women, however, no overall increased risk for lung cancer was observed. Although not significant, our findings thus indicated an increased risk of lung cancer among men living close to the nonferrous smelter. This increase appeared to concern primarily men exposed during the early years of operations, when emissions were very high. © 2003 Wiley‐Liss, Inc.
ERK inhibitor PD98059 enhances docetaxel‐induced apoptosis of androgen‐independent human prostate cancer cells
Authors: Zelivianski Stanislav , Spellman Matthew , Kellerman Mark , Kakitelashvilli Vladimir , Zhou Xia‐Wei , Lugo Esmeralda , Lee Ming‐Shyue , Taylor Rodney , Davis Thomas L. , Hauke Ralph , Lin Ming‐Fong
Published: September 2003

Anticancer drugs docetaxel and vinorelbine suppress cell growth by altering microtubule assembly and activating the proapoptotic signal pathway. Vinorelbine and docetaxel have been approved for treating several advanced cancers. However, their efficacy in the management of advanced hormone‐refractory prostate cancer remains to be clarified. Microtubule damage by some anticancer drugs can activate the ERK survival pathway, which conversely compromises chemotherapeutic efficacy. We analyzed the effect of ERK inhibitors PD98059 and U0126 on vinorelbine‐ and docetaxel‐induced cell growth suppression of androgen‐independent prostate cancer cells. In androgen‐independent C‐81 LNCaP cells, inhibition of ERK by PD98059, but not U0126, plus docetaxel resulted in enhanced growth suppression by an additional 20% compared to the sum of each agent alone (p < 0.02). The combination treatment of docetaxel plus PD98059 also increased cellular apoptosis, which was in part due to the inactivation of Bcl‐2 by increasing phosphorylated Bcl‐2 by more than 6‐fold and Bax expression by 3‐fold over each agent alone. At these dosages, docetaxel alone caused only marginal phosphorylation of Bcl‐2 (10%). Docetaxel plus U0126 had only 20% added effect on Bcl‐2 phosphorylation compared to docetaxel alone. Nevertheless, both U0126 and PD98059 exhibited an enhanced effect on docetaxel‐induced growth suppression in PC‐3 cells. No enhanced effect was observed for vinorelbine plus PD98059 or U0126. Thus, the combination therapy of docetaxel plus PD98059 may represent a new anticancer strategy, requiring lower drug dosages compared to docetaxel monotherapy. This may lower the cytotoxicity and enhance tumor suppression in vivo. This finding of a combination effect could be of potential clinical importance in treating hormone‐refractory prostate cancer. © 2003 Wiley‐Liss, Inc.
Long‐term cancer pain management in morphine pre‐treated and opioid naive patients with transdermal fentanyl
Authors: Mystakidou Kyriaki , Tsilika Eleni , Parpa Efi , Kouloulias Vassilios , Kouvaris Ioannis , Georgaki Stavroula , Vlahos Lambros
Published: September 2003

There is emerging data supporting the use of TTS‐F (transdermal therapeutic system‐fentanyl) in opioid naive patients. Our study examines the safety and efficacy of TTS‐F in the long‐term control of cancer pain in opioid naive patients and those transferring from oral morphine. Pain was assessed in 589 patients (Group A: 268 opioid naive, Group B: 321 transferring from morphine) using a Visual Analogue Scale (VAS; 0–10), based on selected questions from the Greek Brief Pain Inventory (GBPI). Overall treatment satisfaction was assessed on a 4‐point scale. Quality of Life (QOL) and ECOG (0–4) status were also recorded. These were assessed in relation to TTS‐F dose, pain type (neuropathic, combined, nociceptive), concomitant use of anti‐inflammatory drugs and other demographic data. Of 589 patients, 59 (10%) withdrew as a result of inadequate pain satisfaction or for other reasons. There were no discontinuations due to side effects; no Grade 3–4 events occurred. A total of 530 continued on‐study, 211 patients died during study period and 295 departed; all (506; 89%) were satisfied with their pain relief. Analysis of patients at baseline, 28 days, 6 and 12 month time points (n = 153 Group A; n = 214 Group B) with respect to QOL and pain measures indicated a statistically significant (p < 0.001) improvement in all measures across time independent of pain type, or any other patient characteristic(s). In patients with intolerable pain, transfer to TTS‐F offers an efficient and safe long‐term analgesic option. TTS‐F offers durable long‐term maintenance of pain relief with acceptable side effects in opioid naive patients. In general, TTS‐F as a first line analgesic approach for carefully selected and monitored patients experiencing moderate to severe cancer pain should be considered. © 2003 Wiley‐Liss, Inc.
High frequency of activated K‐ras codon 15 mutant in colorectal carcinomas from Taiwanese patients
Authors: Wang Jaw‐Yuan , Hsieh Jan‐Sing , Chen Fang‐Ming , Yeh Ching‐Sheng , Alexandersen Ketil , Huang Tsung‐Jen , Chen David c.p. , Lin Shiu‐Ru
Published: September 2003

Colorectal carcinogenesis is regarded as a multistep process resulting from accumulation of genetic alterations, including activation of protooncogenes and inactivation of tumor suppressor genes via signal transduction trigger the stage‐wise progression to malignancy. The reported incidence of K‐ras mutation detected in general tissue samples ranges from 21–60% in primary colorectal cancers (CRC). To assess the prevalence and spectrum of K‐ras mutations in Taiwanese patients with CRC, we analyzed 65 CRC patients by polymerase chain reaction‐single strand conformation polymorphism analysis, followed by direct sequencing. K‐ras mutations were detected in 43.1% (28 of 65) of the tumors. The mutational hot spots were located at codons 12, 13, 15 and 20, especially with the highest frequency at codon 15. To understand whether the codon 15 mutations in CRC were associated with activation of K‐ras oncogene and the alterations of its biocharacteristics, mutant K‐ras genes were cloned from tumor tissues and then inserted into expression vector pBKCMV to construct the prokaryotic expression plasmid pK15MCMV. Mutant K‐ras genes were expressed at high levels in E. coli and the mutant K‐ras proteins were shown to be functional with respect to their well‐known specific, high‐affinity, GDP/GTP binding. The purified K‐ras protein from E. coli was then measured for its intrinsic GTPase activity and the extrinsic GTPase activity in the presence of GTPase‐activating protein for ras. We found that the extrinsic GTPase activity of the codon 15 mutant K‐ras proteins (p21K‐ras15M) in the presence of GAP is much lower than that of the wild‐type K‐ras protein (p21 BN), whereas the intrinsic GTPase activity is nearly the same as that of the wild‐type K‐ras protein. The results indicated that mutation at the codon 15 of K‐ras gene indeed decreased GTPase activity in CRC, however, its association with tumorigenesis of CRC needs be clarified by further studies. © 2003 Wiley‐Liss, Inc.
Magnitude and polarization of P53‐specific T‐helper immunity in connection to leukocyte infiltration of colorectal tumors
Authors: van der Burg Sjoerd H. , Menon Anand G. , Redeker Anke , Franken Kees L.M.C. , Drijfhout Jan W. , Tollenaar Rob A.E.M. , Hartgrink Henk H. , van de Velde Cornelis J.H. , Kuppen Peter J.K. , Melief Cornelis J.M. , Offringa Rienk
Published: September 2003

The tumor antigen p53 is mutated frequently and overexpressed in colorectal cancer. As a result, patients with this type of cancer commonly display p53‐specific T‐helper (Th) immunity. Examination of the cytokines produced by these Th‐cells showed that a majority of the proliferative p53‐specific T cell cultures produced none of the key cytokines (IFNγ, TNFα, IL‐4, IL‐5 or IL‐10), indicating that these p53‐specific Th‐responses are not polarized. In patients who exhibited p53‐specific reactivity against multiple p53‐epitopes, non‐polarized responses could be found side by side with polarized Th‐responses that produced INFγ or other cytokines such as IL‐10. Patients who exhibited p53‐specific IFNγ‐producing Th cell‐immunity before surgical excision of the tumor displayed higher numbers of tumor infiltrating intraepithelial leukocytes (p = 0.04) than patients lacking such responses, suggesting that the systemic presence of p53‐specific Th‐cells positively affects local tumor‐immunity. Our data concerning the polarization‐state of p53‐specific Th immunity in colorectal cancer patients support the use of vaccine formulations that induce strong Th1‐polarized p53‐specific immunity to ensure proper (re‐)programming of the anti‐tumor response. © 2003 Wiley‐Liss, Inc.
Mapping of candidate region for chordoma development to 1p36.13 by LOH analysis
Authors: Riva Paola , Crosti Francesca , Orzan Francesca , Dalprà Leda , Mortini Pietro , Parafioriti Antonina , Pollo Bianca , Fuhrman Conti Anna Maria , Miozzo Monica , Larizza Lidia
Published: September 2003

Various cytogenetic and molecular findings indicate 1p36 loss as a consistent change in sporadic and inherited chordoma, a rare embryogenetic neoplasm arising from notochord remnants. We studied 27 sporadic chordomas by means of loss of heterozygosity (LOH) of 31 microsatellites localized to the 1p36.32–36.11 region, and restricted the minimal LOH interval shared by 85% of the tumours to 1p36.13. We also used RT‐PCR analysis to investigate the role of the candidate genes CASP9, EPH2A, PAX7, DAN and DVL1, which were selected on the basis of the physical mapping of the LOH region and their plausible oncosuppressor function. RT‐PCR analysis showed the presence of DAN and PAX7 transcript fragments of the expected size in all of 8 chordoma samples, whereas the CASP9‐specific fragment was observed in only 3 and EPH2A was absent in one. Smaller than expected DVL1 transcripts were found in 4 tumours as well as in their normal counterpart (nucleus pulposus), which also showed a typically sized transcript. Sequencing revealed the skipping of 3 exons in the smallest DVL1 fragment, thus leading to a frameshift and predicting a truncated DVL1 gene product. Our study of the largest cohort of chordoma patients recruited so far indicates a common molecular lesion at 1p36.13, and suggests that the CASP9, EPH2A and DVL1 genes may play an onco‐suppressing role and be involved in the development of chordoma. © 2003 Wiley‐Liss, Inc.
Versican in epithelial ovarian cancer: Relation to hyaluronan, clinicopathologic factors and prognosis
Authors: Voutilainen Kirsi , Anttila Maarit , Sillanpää Sari , Tammi Raija , Tammi Markku , Saarikoski Seppo , Kosma Veli‐Matti
Published: September 2003

Versican, a proteoglycan previously reported to increase in other malignant tumours, was studied immunohistochemically in 299 primary epithelial ovarian cancers, their 43 metastases and 6 normal ovaries to evaluate its prognostic value and relation to hyaluronan, another extracellular matrix molecule increased in cancer and a binding partner of versican. The stainings were scored according to the area percentage of strong versican signal of total peri‐ and intratumoural stroma as low (<15%) or high (≥15%). Epithelial staining of the tumours was scored as positive or negative. Low and high area percentage of strong stromal versican staining were observed in 133 and 166 carcinomas, respectively. A low area percentage of strong stromal versican staining correlated with mucinous histology (p= 0.019) and early International Federation of Gynecologists and Obstetritians (FIGO) stage (p< 0.0005), whereas a high percentage was associated with reduced 5‐year survival rate of the patients (44%vs.32%;p= 0.032). Versican was associated with the cancer cells in 151 tumours and correlated with clear cell histology (p< 0.0005), early FIGO stage (p= 0.049) and increased recurrence‐free survival (63%vs.47%;p= 0.032). However, in Cox's multivariate analyses with the conventional prognostic factors included, neither stromal nor cancer cell‐associated versican reached a significant prognostic value. Versican is thus enriched in the malignant stroma surrounding and promoting the growth of ovarian cancer, probably acting with hyaluronan, and associates with unfavourable prognosis but does not constitute an independent indicator of patient survival. © 2003 Wiley‐Liss, Inc.
Derivation and characterization of a Wilms' tumour cell line, WiT 49
Authors: Alami Jennifer , Williams Bryan R. , Yeger Herman
Published: September 2003

Wilms' tumour is a pediatric neoplasm exhibiting histologic features of developing kidney. Although the majority of Wilms' tumour patients are treated effectively, approximately 15% develop metastases and of these, 30% succumb to their disease. The biologic factors governing Wilms' tumour metastasis are largely unknown. Attempts at deriving representative Wilms' tumour cell lines, which could facilitate functional studies, have only been partially successful thus far. We now report on derivation and characterization of a Wilms' tumour cell line, WiT 49, from a first‐generation xenograft of a human Wilms' tumour lung metastasis. WiT 49 recapitulates the phenotype of the parent tumours (primary and lung metastasis) and expresses normal WT1, overexpresses IGFII and carries a frequently identified p53 mutation. We recently reported overexpression of hepatocyte growth factor(HGF) and its receptor met in a series of Wilms' tumours with higher levels in homotypic metastatic cases. We therefore examined WiT 49 for expression of HGF/met and for met signaling targets associated with cell adhesion and cytoplasmic mediators of transcription using Western blot, co‐immunoprecipitation, immunofluorescence labeling and zymography. Our results show co‐expression of HGF and met protein, absence of E‐cadherin, high levels of beta‐catenin co‐immunolocalized to met at the cell membrane and moderate levels of gamma‐catenin and ezrin protein expression. After cell fractionation, beta‐catenin was detected in the cytoplasm and nuclei of WiT 49 with relatively higher levels in the cytoplasm as compared to nuclei. Examination of MMP expression in WiT 49 showed constitutive activation of MMP 9 and latent MMP 2 supporting possible beta‐catenin‐mediated transcriptional activation. The WiT 49 cell line responded to recombinant human HGF by an increase in the expression of the met receptor, recruitment of the Gab‐1 adapter protein to met and release of bound beta‐catenin from met. Our studies therefore establish WiT 49 as a representative Wilms' tumour cell line derived from a lung metastasis that co‐expresses HGF/met and shows absence of the cadherin‐catenin complex supporting a role for these factors in regulation of the invasive and metastatic phenotype in Wilms' tumour. © 2003 Wiley‐Liss, Inc.
Association between NAD(P)H: Quinone oxidoreductase 1 (NQ01) inactivating C609T polymorphism and adenocarcinoma of the upper gastrointestinal tract
Authors: Sarbia Mario , Bitzer Monika , Siegel David , Ross David , Schulz Wolfgang A. , Zotz Rainer B. , Kiel Sybille , Geddert Helene , Kandemir Yasemin , Walter Alexandra , Willers Reinhart , Gabbert Helmut E.
Published: September 2003

NQO1 is an antioxidant enzyme, important in the detoxification of environmental carcinogens. A single nucleotide polymorphism (C→T) at position 609 of the NQO1 cDNA has been associated with susceptibility to tumours induced by chemical carcinogens. In our case‐control study, we determined the prevalence of the C609T NQO1 polymorphism by PCR‐RFLP analysis in Caucasian patients with oesophageal adenocarcinoma (OAC; n=61), cardiac adenocarcinoma (CAC; n=120) or gastric adenocarcinoma (GAC; n=203) vs. a control group that consisted of 252 healthy blood donors. Additionally, NQO1 mRNA expression and NQO1 protein expression were determined by RT‐PCR and immunohistochemistry in a subset of cases. The NQO1 C609T genotype distribution was significantly different among controls (C/C, 73.4%; C/T, 25.0%; T/T, 1.6%) as compared to OAC patients (C/C, 49.2%; C/T, 47.5%; T/T, 3.3%; p=0.0004), CAC patients (C/C, 55.8%; C/T, 40.0%; T/T, 4.2%; p=0.0005) and with GAC patients (C/C, 65.5%; C/T; 30.6%, T/T; 3.9%; p=0.0377). The 609T allele overall frequency was 0.141 in controls, 0.270 in OAC patients, 0.241 in CAC patients and 0.192 in GAC patients. Individuals carrying 1 or 2 609T alleles had a 2.85‐fold higher risk (95% CI: 1.61–5.07; p=0.0003) for the development of OAC and a 2.18‐fold higher risk (95% CI: 1.38–3.44; p=0.0007) for the development of CAC than wild‐type gene homozygotes. Immunohistochemical analysis showed NQO1 protein expression in 133 carcinomas, whereas 17 carcinomas were negative. Negativity for NQO1 protein expression correlated strongly with the NQO1 genotype being present in 3.9% of cases with C/C, 13.9% of cases with C/T and 62.5% of cases with T/T genotype (p<0.001). In contrast, NQO1 mRNA expression was detectable irrespective of underlying genotype. In conclusion, determination of the NQO1 genotype may gain importance as a stratification marker in future prevention trials for adenocarcinoma of upper gastrointestinal tract. © 2003 Wiley‐Liss, Inc.
Human papillomavirus type 16 and 18 L1 protein peptide binding to VERO and HeLa cells inhibits their VLPs binding
Authors: Vera‐Bravo Ricardo , Ocampo Marisol , Urquiza Mauricio , García Javier E. , Rodríguez Luis E. , Puentes Alvaro , López Ramses , Curtidor Hernando , Suárez Jorge E. , Torres Elizabeth , Guzmán Fanny , Díaz Diana , Cortes Jimena , Bravo María M. , Cómbita Alba L. , Orozco Oscar , Patarroyo Manuel E.
Published: September 2003

Human papillomaviruses (HPVs) are the cause of epithelial lesions, HPV type 16 and type 18 being associated with the development of anogenital cancer. The L1 Major Capsid Protein (L1) represents about 90% of total HPV protein and is involved in virus‐host cell interaction, but little is known about this binding process. L1 sequences from HPV types 16 and 18 were synthesized in 56 20‐mer peptides, covering the entire protein, HPLC‐purified,125I‐radiolabeled and tested in VERO and HeLa cell‐binding assays to identify those peptides with high specific binding activity. Peptides 18283 (residues 54–77) and 18294 (274–308) from HPV16 L1, as well as 18312 (59–78) and 18322 (259–278) from HPV18 L1, presented high specific target cell binding activity. Peptide 18283 and 18294 affinity constants were 300 and 600 nM, respectively. Enzyme cell treatment before binding assay indicated that VERO and HeLa cell peptide receptor is a surface‐exposed protein. There was a 60% reduction in peptide 18283 binding to heparin lyase‐treated cells. Cross‐linking assays showed that these proteins molecular weights were around 69 and 54 kDa. Peptides 18283 and 18294 specifically inhibited HPV‐16 VLP binding to HeLa cells. According to the L1‐ and VLP‐reported structure, both peptides are close on the VLP‐surface, belonging to the outer surface broad pockets suggested as being potential receptor sites. Furthermore, it has been reported that a conserved motif from peptide 18294 is the target for neutralizing antibodies. These results suggest that such binding sequences are used by the virus as cell‐binding regions. © 2003 Wiley‐Liss, Inc.
Cyclopalladated compounds as chemotherapeutic agents: Antitumor activity against a murine melanoma cell line
Authors: Rodrigues Elaine G. , Silva Luiz S. , Fausto Daniela M. , Hayashi Marina S. , Dreher Simone , Santos Edson L. , Pesquero João B. , Travassos Luiz R. , Caires Antonio C.F.
Published: September 2003

Palladacycle compounds obtained from N, N‐dimethyl‐1‐phenethylamine (dmpa), phenyl‐2‐pyridinyl‐acetylene and 1‐phenyl‐3‐N, N‐dimethylamine‐propine, respectively, were complexed to 1, 2 ethanebis (diphenylphosphine) (dppe) ligand to synthesize antitumor cyclopalladated complexes that were tested in vitro and in vivo against syngeneic B16F10‐Nex2 murine melanoma cells of low immunogenicity implanted subcutaneously in mice. Complexes were not toxic to mice injected 3 times i.p. with as much as 60 μM/animal/week. Of 3 cyclopalladated complexes that were inhibitory in vitro at low concentrations (<1.25 μM), complex 7a was the most active in vivo, delaying tumor growth and prolonging animal survival. In vitro, binucleate complex 7a caused a collapse of respiratory activity with an abrupt decrease of extracellular acidification on short incubation (up to 100 min), followed by DNA degradation after 24 hr. The apoptosis‐like reaction to this Pd‐complex was not accompanied by increased levels of caspases 1 and 3. Complex 7a bound to a bacterial plasmid DNA, causing late conformational changes after 24 hr. Two other complexes with different C, N‐cycles were also apoptotic and 2 binucleated ones were inactive. These results introduce the palladacycle‐dppe complexes as promising antitumor drugs with exquisite structural specificities and for action in vivo and in vitro. © 2003 Wiley‐Liss, Inc.
Hepatic stellate cell activation in vitro: Cell cycle arrest at G2/M and modification of cell motility
Authors: da Silva Fabiana M. , Guimarães Eduardo L.M. , Grivicich Ivana , Trindade Vera Maria T. , Guaragna Regina Maria , Borojevic Radovan , Guma Fátima C.R.
Published: September 2003

Hepatic fibrosis is a common response to chronic liver injury and is characterized by increased production of extracellular matrix components, whose major part is produced by hepatic stellate cells activated by inflammatory mediators to proliferate and migrate into the injured regions. GRX cells are a model of hepatic stellate cells characterized as myofibroblasts by morphological and biochemical criteria. We have recently shown that they respond to inflammatory mediators and cytokines present in the concanavalin A‐activated spleen cell supernatant (SCS) by quantitative changes in the expression of intermediate filaments. The present study investigated the effects of SCS and TNF‐α on the GRX cell proliferation and on the organization of the actin cytoskeleton. SCS and TNF‐α diminished the culture cell density, with an increase of cell [3H]thymidine incorporation and of cellular protein content, indicating an arrest in the G2/M phase of the cell cycle, which was reversible 48 h after removal of SCS. This effect was abrogated by dibutiryl‐cAMP. Actin cytoskeleton reorganization was observed after 24 h treatment, indicating increased cell motility. Our results suggest that inflammation‐dependent activation of stellate cells occurs in ordered interaction and coordination of proinflammatory agents. The increase of cAMP levels activates the conversion of lipocytes into myofibroblasts and increases the number of cells that can participate in repair. Since cAMP retains cells in the G1 phase, cytokines of the TNF‐α group are required for cell proliferation inducing the entry into the S phase. The progression through the G2/M checkpoint is mediated again by increased cAMP levels. J. Cell. Biochem. 90: 387–396, 2003. © 2003 Wiley‐Liss, Inc.
Method of platinum‐carbon coating of ultrathin sections for transmission and scanning electron microscopy: An application for study of biological composites
Authors: Schwarz H. , Gorb S.
Published: September 2003

Many biological materials are composites containing two or more components with different mechanical properties. This study is concerned with the application of a method of platinum‐carbon coating (Pt/C) of ultrathin sections for TEM and SEM studies of the design of natural composite materials. The changes in profile of the ultrathin resin‐embedded sections during different stages of the preparation reflect the material properties of the various components: stiffer regions deform less than softer ones. Such changes in the section profile can be visualized by the Pt/C method and used as evidence of specific material properties in particular regions of composite materials. The method increases the relief contrast, improves the 3D‐view of structures, and in combination with standard TEM and SEM procedures can provide clear demonstrations of material design. The distribution of chitin crystallites in the insect cuticle and the ultrastructure of the pore canal system specialized for the transport of epidermal secretions to the cuticle surface were studied here as examples. Microsc. Res. Tech. 62:218–224, 2003. © 2003 Wiley‐Liss, Inc.
Technique to study three‐dimensional spatial arrangement of synaptic vesicles using data from single sections
Authors: Nikonenko Alexander G.
Published: September 2003

Synaptic vesicles are membrane‐bound organelles storing neurotransmitters in presynaptic terminals and releasing them into the synaptic cleft. Coordinated movements of synaptic vesicles relate to synaptic function and their spatial arrangement can provide useful information about the activity of a synapse. This article presents a technique to extract quantitative information about three‐dimensional (3D) spatial arrangement of synaptic vesicles from measurements performed on single ultrathin random sections of a presynaptic terminal. The technique presumes quantification of a 2D density as well as 2D spatial pattern formed by vesicle profiles using a minimum spanning tree (MST) algorithm, in digitized micrographs of a presynaptic terminal. Further, original software was used to simulate a 3D spatial arrangement of synaptic vesicles and their random sectioning. A 3D density and pattern of synaptic vesicles were used as basic input parameters of the model, while a 2D density and MST quantities for vesicle profiles served as output, model‐derived parameters allowing one to compare and fit simulated distributions to experimental ones. Pilot simulations performed to check the validity of the technique have shown that a 2D density and MST quantities of vesicle profiles closely relate to a 3D density and spatial pattern of vesicles. The technique was demonstrated in the analysis of spatial distribution of synaptic vesicles in axonal terminals forming asymmetric synaptic densities in the stratum radiatum of the CA1 subfield of the murine hippocampus. Microsc. Res. Tech. 62:201–210, 2003. © 2003 Wiley‐Liss, Inc.
In vitro and in situ experimental model for X‐ray microanalysis of intestinal epithelium
Authors: Vanthanouvong Viengphet , Högman Marieann , Roomans Godfried M.
Published: September 2003

Intestinal chloride (Cl) transport is disturbed in a number of diseases. X‐ray microanalysis can be used to study the distribution of Cl and other ions in intestinal epithelial cells. In this study it was attempted to establish an experimental system that retains the in vivo elemental composition of intestinal epithelial cells. An in vitro system was set up in which a segment of rat intestine was mounted in a bath and perfused with different fluids. The chloride in the bath or in the perfusion fluid could be exchanged for gluconate or bromide to determine the direction of chloride fluxes. An in situ system was set up in which the animal was anesthetized and a segment of the intestine was perfused with different solutions. In the in vitro experiments the concentration of Na and Cl in the epithelial cells increased and that of K decreased. These changes occurred within the first 30 minutes of incubation. Uptake of chloride occurred mainly from the bath, as seen in experiments where bromide was used as a chloride analog. The concentration gradient between bath and tissue determined the extent of chloride uptake. Addition of glucose to the perfusion fluid and bath did not improve the results. In the in situ system, preservation of the intracellular ion composition was better. Acceptable results were obtained with perfusion with Krebs‐Ringer's buffer without glucose for 30 minutes. In this case, the elemental content of the cell did not change appreciably during incubation. If glucose was added, the Na concentration increased in comparison to the control, both in crypt and villus cells. It is concluded that the intestinal epithelium is a sensitive system, very prone to disturbance of its homeostasis. However, the in situ system can be used in studies of agonist‐induced ion transport. Microsc. Res. Tech. 62:211–217, 2003. © 2003 Wiley‐Liss, Inc.
Junctional complex revisited by high‐resolution scanning electron microscopy
Authors: Testa Riva Francesca , Serreli Silvia , Loy Francesco , Riva Alessandro
Published: September 2003

The present study correlates the ultrastructural morphology of junctional complexes as revealed by transmission electron microscopy (TEM) with that observed by high‐resolution scanning electron microscopy (HRSEM), thanks to a new modification of the osmium tetroxide maceration technique. The removal of all cytoplasmic organelles by this technique allows the inspection of the inner side of the plasmalemma. With this treatment, a continuous band of tightly packed particles is observed at the most apical portion of lateral membranes. Just below this band, irregular clusters of apparently identical particles are placed all around the cellular contour. The topographical correspondence among these clusters and spot desmosomes seen by TEM identifies them as desmosomes. The continuous band seems to represent the combination of both zonulae, occludens and adherens. Regarding the nature of the particles, we suppose that they probably consist of peripheral membrane proteins clustered at the cytoplasmic surface of intercellular junctions and involved in the linkage between cytoskeleton and plasmalemma. Microsc. Res. Tech. 62:225–231, 2003. © 2003 Wiley‐Liss, Inc.