bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic alterations of the putative envelope proteins encoding region of the hepatitis C virus in the progression to relapsed phase from acute hepatitis: Humoral immune response to hypervariable region 1
Authors: Sekiya Hitomi , Kato Nobuyuki , Ootsuyama Yuko , Nakazawa Takahide , Yamauchi Katsumi , Shimotohno Kunitada
Published: February 2007

Hypervariable region I (HVRI) of the putative second envelope glycoprotein (gp70) of hepatitis C virus (HCV) undergoes sequential alterations at intervals of several months during the chronic phase of hepatitis. To evaluate the implications of sequence variability in HVRI of HCV, we investigated the sequence variability of the whole envelope‐protein(gp35 and gp70)‐coding regions of HCV genome derived from patient M in acute and relapsed phases (8‐month interval) of hepatitis. From this analysis, we found that a Leu (position 405) in HVRI substituted to Pro, and that 4 additional substitutions could be detected in gp70 during the relapsed phase. Sequence‐specific antibody against HVRI derived from patient M was first detected in the serum at 8 months after the onset of hepatitis, but no other specific antibodies against peptides containing aminoacid position(s) substituted in regions other than HVRI could be detected. Epitope mapping using the sequence of HVRI derived from the acute phase of hepatitis was also performed, and a B‐cell epitope (positions 397 to 407) of 11 amino acids was identified. However, the Pro variant at position 405 did not display an escape pattern from the antibody produced at 8 months after the onset. In addition, we demonstrated the existence of important a mi no‐acid residue positions which are recognized by the anti‐HVRI antibody produced in patient M using introduction point mutations within HVRI.
Neuroblastoma cell detection by reverse transcriptase‐polymerase chain reaction (RT‐PCR) for tyrosine hydroxylase mRNA
Authors: Burchill S. A. , Bradbury F. M. , Smith B. , Lewis I. J. , Selby P.
Published: February 2007

The presence of tumour cells in peripheral blood of neuroblastoma patients is of considerable clinical importance. Nucleic acid amplification offers an opportunity to detect very small numbers of such cells, but in neuroblastoma a frequent specific abnormality in the tumour DNA suitable for this purpose has yet Co be identified. To facilitate the detection of such cells we have developed RT‐PCR using tyrosine hydroxylase (TH) as a tissue‐specific target gene. TH mRNA was detected in 3 neuroblastoma cell lines and in all neuroblastoma tumours examined, but was undetectable in peripheral blood from children without neuroblastoma. The method was highly sensitive, detecting 1‐10 neuroblastoma cells per 107 blood cells. Thirty blood samples from 24 patients were analysed and results were compared with known disease status. At diagnosis 4/7 patient blood specimens were positive; the four positive samples were from stage‐4 patients. In blood samples from these patients 6‐8 weeks after the initiation of treatment, TH mRNA was undetectable. Of 7 samples taken at the time of clinical relapse, 5 were positive; 4 of these were from patients with evidence of disseminating disease. Of 16 blood samples from disease‐free patients, 14 were negative and 2 were positive. One positive patient in this group subsequently had a clinical relapse. These results show that this technique is of value for detecting neuroblastoma cells in peripheral blood. The significance of these cells at diagnosis, during treatment or on follow‐up requires further evaluation.
Deletion mapping of chromosome 19 in human gliomas
Authors: von Deimling Andreas , Nagel Judith , Bender Bernhard , Lenartz Doris , Schramm Johannes , Louis David N. , Wiestler Otmar D.
Published: February 2007

There is evidence that a putative glioma tumor suppressor locus resides on the long arm of chromosome 19. We present data on 161 gliomas from IS6 patients, which were studied by microsatellite analysis for loss of heterozygosity (LOH) on chromosome 19. Eight loci on the long arm and 2 loci on the short arm of chromosome IV were examined. LOH on I9qwas observed in 3/19 astrocytomas (WHO grade II), 12/27 anaplastic astrocytomas (WHO grade III), 16/76 cases of glioblastoma multiforme WHO (grade IV), 4/9 oligodendrogliomas (WHO grade II), 3/5 anaplastic oligodendrogliomas (WHO grade III), 5/9 mixed oligo‐astrocytomas (WHO grade II) and 8/10 anaplastic oligo‐astrocytomas (WHO grade III). While 31 of the tumors with LOH on chromosomal arm I9q exhibited allelic loss at every informative locus, 20 tumors showed terminal or interstitial deletions. In contrast to astrocytomas and glioblastomas, tumors with an oligodendroglial component had predominantly lost the entire long arm of chromosome 19. The common region of overlap in gliomas was located on 19q 13.2‐q 13.4 between the markers D 19S 178 and D 19S 180. Our data confirm the involvement of a putative tumor suppressor gene on chromosomal arm 19q in gliomas and assign this gene to 19q 13.2‐q 13.4.
Incidence of breast cancer in women with fibroadenoma
Authors: Levi Fabio , Randimrison Lalao , Te Van‐Cong , la Vecchia Carlo
Published: February 2007

There is a general agreement that fibroadenoma is associated with a certain increase in breast cancer risk, but the risk estimates appear heterogeneous according to various studies. To provide further quantitative and population‐based estimates of breast cancer risk in women with histologically confirmed fibroadenomas, we linked data of 1,461 incident cases of fibroadenoma (median age, 37 years) to the Cancer Registry of the Swiss Canton of Vaud (about 600,000 inhabitants) over the period 1977–1991. After exclusion of 57 synchronous breast neoplasms, a total of 17 cases of breast cancer were observed vs. 10.9 expected (standardised incidence ratio, SIR, 1.6; 90% confidence interval: 1.1‐2.1). The SIR was similar in the short term (3–5 years) and in the medium‐long term (>5 years) following diagnosis of fibroadenoma. The cumulative risk of invasive breast cancer was 0.7% after 5 years and 2.2% after 12 years following diagnosis of fibroadenoma. This is not negligible on account of the young age of our cohort.
Characterization of a bombesin/gastrin‐releasing peptide receptor on a human gastric‐cancer cell line
Authors: Preston Shaun R. , Woodhouse Linda F. , Gokhale Jay , Miller Glenn V. , Primrose John N.
Published: February 2007

This study examined the expression of receptors of the bombesin (BBS) family in human gastric‐cancer cell lines. Of 5 cell lines screened, only one, St42, demonstrated specific binding sites for 125I‐Tyr4‐BBS, which have been further characterized. This binding was saturable, and temperature‐ and time‐dependent. Scatchard analysis of displacement data performed at 37°C revealed 2 binding sites: a high‐affinity, low‐capacity site (KD = 0.13 nM, Bmax = 1500 sites/cell) and a lower‐affinity, higher‐capacity site (KD = 11 nM, Bmax = 35,000 sites/cell); the latter was lost when internalization of peptide was prevented, suggesting that it may be an artefact. Displacement assays with gastrin‐releasing peptide (GRP) and neuromedin B (NMB) revealed that the receptor was of the GRP‐preferring sub‐type (GRP IC50 = 0.35 nM; NMB IC50 = 112 nM). Co‐valent cross‐linking of 125I‐Tyr4‐BBS to the receptor demonstrated the presence of a single band corresponding to a molecular weight of 37 to 44 kDa on SDS‐PAGE, similar to that of the cloned GRP receptor protein core. G‐protein linkage of this receptor was demonstrated by selective inhibition of 125I‐Tyr4‐BBS binding by guanosine nucleotides. The binding of BBS to the receptor resulted in a rise in intracellular calcium. Three of four structurally distinct BBS antagonists bound to the receptor with high affinity, but [DPhe12, Leu14]‐bombesin did not cause any displacement of 125I‐Tyr4‐BBS even at 10 mM. The functional significance of GRP receptors on human gastric‐cancer cells is as yet unknown, but further studies may determine whether such receptors have importance in the therapy of gastric cancer.
Isolation and characterization of tumor‐infiltrating lymphocytes from cervical carcinoma
Authors: Hilders Carina G. J. M. , Ras Liesbeth , van Eendenburg Jaap D. H. , Nooyen Yvonne , Fleuren Gert Jan
Published: February 2007

The evidence that virus‐induced tumors generally elicit T‐cell responses prompts the notion that HPV‐related cervical carcinoma would be amenable to treatment by T‐cell‐mediated adoptive therapy. Therefore, we cultured and cloned tumorinfiltrating lymphocytes (TIL) from a patient with cervical carcinoma and studied the in vitro characteristics of these TIL by using the established autologous tumor‐cell line. After stimulation of bulk TIL cultures with 1,000 Units/ml recombinant interleukin 2 (rIL‐2), followed by limiting dilution, T‐cell clones were generated in the presence of 20 U/ml rIL‐2 and irradiated autologous tumor cells, PBLs and EBV‐transformed B‐cell lines. Phenotypically, all clones were CD3/CD8‐positive with a heterogeneous CD56 expression. All expressed preferential cytolytic activity against autologous tumor cells, did not lyse autologous lymphoblasts, and were cytotoxic against the NK‐sensitive cell line K562. A minor lytic capacity was detectable on allogeneic cervical tumor‐cell lines or tumor‐cell lines of other histologic types. Cytotoxicity against the autologous tumor could be inhibited by anti‐CD3, anti‐CD8 and anti‐ICAMI but not by anti‐HLA class‐1 (W6/32, B9.12.I), anti‐allele‐specific HLA determinants and anti‐LFA‐3 antibodies. We demonstrate a highly specific autologous lytic activity of cervical carcinoma TIL, in which a CD3‐associated surface antigen recognition is involved. These results may prove useful in further studies on adoptive immunotherapy of cervical cancer patients. © 1994 Wiley‐Liss, Inc.
Cell kinetics in human breast cancer: Comparison between the prognostic value of the cytofluorimetric S‐phase fraction and that of the antibodies to Ki‐67 and PCNA antigens detected by immunocytochemistry
Authors: Gasparini Giampietro , Boracchi Patrizia , Verderio Paolo , Bevilacqua Pierantonio
Published: February 2007

The determination of cell proliferation is one of the more widely used tools for assessing prognosis. However, additional research in this field is warranted because today there are several methodological procedures available for monitoring cell kinetics and it has still not been established which is the most reliable marker of proliferation and which possesses the greatest prognostic value. We performed this study in a series of primary invasive breast cancers to compare the prognostic value of S‐phase fraction (SPF) by flow cytometry, the most widely used method for detecting proliferation at present, with that of antibodies to Ki‐67 and PC‐10 to proliferating‐cell nuclear antigen (PCNA) detected by immunocytochemical methods. A significant linear relationship was observed only between SPF and Ki‐67. In univariate analysis SPF and Ki‐67 values, nodal status, histological grading and peritumoral lymphatic‐vessel invasion were significant predictors of relapse‐free survival (RFS). As far as overall survival (OS) is concerned, only SPF, Ki‐67 and nodal status were significantly associated with the risk of death. PCNA had no prognostic value for either RFS or OS. In multivariate analysis only SPF and nodal status retained a significant and independent prognostic value. Neither the cell‐kinetics parameters assessed by immunocytochemistry (i. e. Ki‐67 and PCNA) nor histological grading were independent prognosticators. In conclusion, our results provide evidence that the determination of SPF by flow cytometry was the strongest cell‐kinetics marker used to assess prognosis in this series of breast cancers. However, different and novel markers of cell kinetics need to be compared in larger series in order to identify the best one. © 1994 Wiley‐Liss, Inc.
Phorbol ester induced rapid attachment and spreading of melanoma cells and the role of extracellular matrix proteins
Authors: van Leeuwen Robert L. , Dekker Sybren K. , Arbiser Jack L. , Vermeer Bert Jan , Bruijn Jan A. , Byers H. Randolph
Published: February 2007

Phorbol 12‐myristate 13‐acetate (PMA) is a tumour promotor that acts as a potent protein kinase C (PKC) activator that has significant effects on tumour cell attachment and spreading. We tested whether these effects of PMA may be observed in human melanoma cells, and whether a specific response to extracellular matrix proteins may be mediated by shifts in the expression of β1 integrins. We used cell attachment assays, video time lapse cell spreading assays, flow cytometry, function blocking monoclonal antibodies (MAbs) and PKC inhibitor Calphostin C to address these questions. We established that PMA induces a rapid and temporary enhancement of cell attachment and spreading which was not accompanied by a significant change in the expression of β1 integrins. Spreading of melanoma cells that were not stimulated with PMA could be significantly blocked with a function blocking MAb (clone P4C10) against the common β1 integrin subunit. The spreading and attachment of the PMA treated cells was also significantly reduced, but less so, after MAb treatment. The PMA enhanced cell attachment and spreading could be effectively blocked by RGD sequences and PKC inhibitor. Taken together, our data indicate that PMA induces a rapid and temporary ECM‐dependent enhancement of melanoma cell attachment and spreading, and that the response to ECM components appears not to be due to significant shifts in β1 integrin expression, but rather to activation of β1 integrins. © 1994 Wiley‐Liss, Inc.
E‐selectin binding by pancreatic tumor cells is inhibited by cancer sera
Authors: Sawada Tetsuji , Ho Jenny J. L. , Chung Yong‐Suk , Sowa Michio , Kim Young S.
Published: February 2007

Tumor cells interact with endothelial cells during both intraand extravasation. Understanding how these interactions are modulated could lead to the development of ways to alter the metastatic potential of tumor cell. Three pancreatic cancer cell lines, SW1990, CAPAN‐2 and PANC‐1, were examined for their ability to bind to the endothelial cell adhesion molecule E‐selectin (ELAM‐1). SW1990 cells exhibited highest binding, highest surface expression of the carbohydrate antigens sialylated Lewis2 (sLe2) and sialylated Lewisx (sLex) and released the most high m. w. sLe2 and sLex antigens. Expression of sLe2 and sLex antigens and binding to E‐selectin were reduced by pre‐treatment of SW1990 cells with the O‐linked glycosylation inhibitor benzyl‐α‐GaINAc but not with the N‐linked glycosylation inhibitor tunicamycin. Expression of peptide epitopes associated with MUCI apomucins was increased by benzyl‐α‐GaINAc. Cell binding was greatly reduced by mucins purified from SW1990 xenografts and by an antibody against sLe2. An antibody against sLex had a much less marked effect. Sera from pancreatic cancer patients reduced SW1990 cell binding to E‐selectin but sera from normals did not. The degree of inhibition was related to the sLex level in the sample. When cancer serum was separated by column chromatography on Sephacryl S‐400, the void volume fractions contained most of the sLe2 and sLex antigens and most of the inhibitory activity to E‐selectin binding. Differences in the relative availability of sLe2 and sLex ligands on serum molecules and on the SW1990 cell surface may account for the differences between antibody and serum inhibition results. Thus SW1990 cell adhesion to E‐selectin is mediated by ligands on mucinous glycoproteins, and adhesion can be inhibited by mucins, high blood levels of sLex and reduction of cellular O‐linked glycosylation. © 1994 Wiley‐Liss, Inc.
Expression of stromelysin‐3 and nm23 in breast carcinoma and related tissues
Authors: Hähnel Erika , Dawkins Hugh , Robbins Peter , Hähnel Roland
Published: February 2007

Biological functions proposed for the ST3 and mm23 genes in tumour development and progression seem to be directly opposed. Stromelysin‐3 (ST3) is a putative member of the matrix metalloproteinase family. ST3 has been implicated in the progression of epithelial malignancies, specifically with regard to an invasive (and therefore potentially metastasizing) phenotype. The nm23 gene, on the other hand, encodes a nucleoside diphosphate kinase which allegedly has a metastasis‐suppressor‐type function. It was therefore of interest to compare the expression of ST3 and nm23 in various surgically excised normal and neoplastic breast tissues. RNA was isolated from over 200 surgical specimens and studied by Northern blots. Normal breast tissues did not express ST3, and ST3 expression was detected in only 1 of 20 normal axillary lymph nodes. None of 7 fibroadenomas expressed ST3. In contrast, 60% of primary and metastatic breast carcinomas contained ST3‐mRNA. The expression of ST3 was mainly confined to invasive carcinomas and was observed less frequently in pure ductal carcinoma in situ (DCIS) lesions. Our results support the suggestion that ST3 expression is related to the malignant process in breast cancer. The role of nm23 is far less clear‐cut.
Simple tandem repeat allelic deletions confirm the preferential loss of distal chromosome 6q in melanoma
Authors: Walker Graeme J. , Palmer Jane M. , Walters Marilyn K. , Nancarrow Derek J. , Parsons Peter G. , Hayward Nicholas K.
Published: February 2007

Karyotypic analysis, loss of somatic heterozygosity, microcell fusion and cDNA transfection studies have provided compelling evidence that at least one tumour suppressor gene for melanoma resides on chromosome 6. In an attempt to further define the regions to which these putative suppressor genes map, we have carried out loss of heterozygosity (LOH) studies on DNA from 25 fresh melanoma tumours for 9 simple tandem repeat (STR) polymorphism markers spanning chromosome 6. Four samples displayed LOH or homozygosity for all markers studied, indicating that they had lost one homologue of chromosome 6. An additional 3 samples showed LOH for all markers on 6q. Furthermore, 30 melanoma cell lines, for which there were no matching somatic DNA samples, were analyzed for hemizygosity of markers on 6q. One cell line had a homozygous deletion of all markers tested and a further 12 cell lines displayed only one allele for 3 or 4 contiguous markers, indicating that most, if not all of these samples were hemizygous for the region of 6q distal to D6S87. Overall, the rate of LOH on 6q in the 55 melanoma DNAs was 35%, and there were no losses of markers on 6p without concomitant loss of markers on 6q. Two of 5 samples derived from primary melanomas showed LOH, which indicates that LOH for the melanoma suppressor gene on 6q, which maps to a region that contains the SOD2 locus, is a frequent and early event in melanoma tumorigenesis.
Prognostic value of deoxyribonucleic acid content in prostate cancer: A review of current results
Authors: Adolfsson Jan
Published: February 2007

A total of 115 articles on prostate cancer were reviewed for data on the prognostic value of DNA content in the tumor cells. In 44 series, data pertinent to this review were found. There was no consensus in the literature with respect to methods of analysis of DNA content or definitions of subclasses of DNA content such as categories of ploidy. The DNA content of prostate cancer cells was strongly related to tumor grade and stage. When analyzed as a single parameter in univariate analyses, the DNA content had a prognostic value with respect to overall or disease‐specific survival. In multivariate analyses the additional prognostic value of the DNA content was less convincing when analysed with tumor grade and stage. The prognostic data from univariate and multivariate analyses available in the literature were mainly derived from patients with advanced disease and data on localized, potentially curable disease were scanty and conflicting.
Stimulation of angiogenesis as an explanation of matrigel‐enhanced tumorigenicity
Authors: Daniel Bonfil R. , Vinyals Antonia , Bustuoabad Oscar D. , Llorens Ana , Benavides Fernando J. , Gonzalez‐Garrigues Milagro , Fabra Angels
Published: February 2007

Matrigel, a reconstituted extract of basement membrane, enhances the growth of different human cancer cell lines when transplanted into nude mice. Here that stimulation was confirmed in the BALB/c murine mammary‐tumor cell line M3MC, as well as in human colon (SW948) and mammary (MDA‐MB‐468) carcinoma cell lines transplanted in nude and SCID mice, respectively. Subcutaneous and intra‐mammary fat‐pad inoculations of Matrigel alone generated an angiogenic response which was macroscopically evident by day 9. Histological analysis of the local host reaction occurring at the site of injection revealed an early peripheral fibroblast response, followed by mononuclear cell infiltration, solid and hollow fibroblast cords projections from the edge to the center of the Matrigel plug, and finally capillary ingrowths. Conditioned media obtained from the gels generated in vivo, acted as very strong chemoattractants for mouse lung capillary endothelial cells, stimulating their motility between 38 and 82 times with respect to the control. Our results suggest an important role of host cells recruited by Matrigel, which could favor angiogenesis of the area and thus facilitate the growth of tumor cells co‐inoculated with the basement membrane extract.
Generation of monoclonal antibodies specific for ganglioside lactones: Evidence of the expression of lactone on human melanoma cells
Authors: Kawashima Ikuo , Kotani Masaharu , Ozawa Hideki , Suzuki Minoru , Tai Tadashi
Published: February 2007

We generated 3 murine monoclonal antibodies (MAbs) specific for ganglioside lactones by immunizing C3H/HeN mice with purified lactones adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. The use of a wide variety of glycolipids, including ganglioside lactones, enabled the precise structures recognized by these MAbs to be elucidated through an ELISA and by immunostaining on thin‐layer chromatography. MAb AMR38, which was generated with GMI lactone, showed restricted specificity, detecting only the GMI lactone used for immunization. None of the other ganglioside lactones, intact gangliosides (including GMI) or neutral glycolipids tested were recognized. In contrast, MAbs AMR40 and AMR19, which were generated with GD Ia lactone and GD3 lactone, respectively, showed broader specificities, recognizing several ganglioside lactones. However, the precise epitopes were different. MAb AMR40 reacted intensely with ganglioside lactones having an external NeuAcα2 → 3Gal‐ sequence (GD Ia, GM3, GM Ib, GT Ib, and IV3Neu´‐nLC4Cer), but not with those having a NeuAcα2 → 8NeuAcα2 → 3Gal‐ sequence. On the other hand, MAb AMR 19 reacted with ganglioside lactones having a NeuAcα2 → 8NeuAcα2 → 3Gal‐ sequence (GD3, O‐Ac‐GD3, GD2, GD Ib, GT Ib, GQ Ib and GT Ia), but not with those having a NeuAcα2 →3Gal‐ sequence. None of the intact gangliosides or neutral glycolipids tested were recognized by the MAbs. We also determined the expression of ganglioside lactones on human melanoma cells grown in athymic nude mice by means of an immunofluorescence technique.
A nonconsensus octamer‐recognition sequence (TAATGARAT‐motif) identifies a novel DNA binding protein in human merkel cell carcinoma cell lines
Authors: Thomson J. A. F. , Leonard J. H. , McGregor K. , Sturm R. A. , Parsons P. G.
Published: February 2007

We have established a number of cell lines from Merkel cell carcinoma (MCC) of the skin. In many respects these cell lines resemble those established from small cell lung cancer (SCLC) and it is difficult to differentiate between metastatic MCC and SCLC on morphological or histochemical criteria. Both are thought to be neuroendocrine tumours and may express a number of neuroendocrine markers. SCLC cell lines express the octamer‐DNA binding transcription factor brn‐2 gene products N‐Oct 3 and N‐Oct 5, which are restricted to the neuroectodermal cell lineage. In DNA binding studies using a consensus octamer recognition site we have found that 4 of 8 MCC cell lines examined expressed brn‐2 in at least trace amounts compared with 3 SCLC cell lines which all expressed brn‐2 proteins at high levels. Moreover, these DNA binding studies were extended by using a high‐affinity brn‐2 recognition site related to the degenerate octamer TAATGARAT‐motif. This identified a novel DNA binding protein in a subset of MCC cell lines. The protein was absent from the three SCLC cell lines, melanoma cells and brain tissue. This binding activity, which we term Merkel cell DNA nuclear factor (MNF), was shown to be specific by competitive inhibition with oligonucleotide binding sites and was not inhibited by polyclonal antisera against the Oct‐I, Oct‐2 or Brn‐2 proteins. This protein may serve as a unique marker for MCC compared with SCLC cells and may be involved in regulating the Merket cell phenotype.
In vivo evidence of the role of α4β1‐VCAM‐1 interaction in sarcoma, but not in carcinoma extravasation
Authors: Paavonen Timo , Tiisala Sinikka , Majuri Marja‐Leena , Böhling Tom , Renkonen Risto
Published: February 2007

Tumor‐cell invasion can occur via either lymphatics or blood vessels. When in the blood circulation, tumor cells have to adhere to endothelium lining the blood vessels before they can extravasate. Several families of adhesion molecules have been recognized: selectins and their oligosaccharide‐containing ligands and integrins and their counter‐receptors belonging to the immunoglobulin superfamily. Besides their essential role in leukocyte extravasation, these adhesion molecules have been proposed by in vitro experiments to be involved in tumor‐cell invasion by facilitating the adhesion of malignant cells to endothelium leading to extravasation and metastasis. We have previously shown that, in vitro, several sarcoma cell lines adhere strongly to cultured endothelial cells via α4β1‐VCAM‐1 interaction. Here we show that sarcoma cells, especially in the metastatic lesions, were strongly α4β1 positive but did not express α4β7, which is another receptor for VCAM‐1. Furthermore, we demonstrate that the capillary endothelium within metastatic sarcoma lesions reacted strongly with anti‐VCAM‐1 antibody and very often the α4β1‐expressing sarcoma cells were localized in the close vicinity of VCAM‐1‐expressing vessels. As control material we analyzed carcinoma specimens, but could not detect any α4‐integrin expression on malignant cells even though the endothelial cells were often VCAM‐1 positive. These results suggest that carcinomas do not use α4β1‐VCAM‐1 in extravasation and, taken together, provide circumstantial evidence that in vitro findings of α4β1‐VCAM‐1‐dependent sarcoma cell adhesion to endothelium can be extended to in vivo situations.
Frequency of cytotoxic T lymphocyte precursors (CTLp) interacting with autologous tumor via the T‐cell receptor: Limiting dilution analysis of specific CTLp in peripheral blood and tumor‐invaded lymph nodes of melanoma patients
Authors: Mazzocchi Arabella , Belli Filiberto , Mascheroni Luigi , Vegetti Claudia , Parmiani Giorgio , Anichini Andrea
Published: February 2007

The frequencies of cytotoxic T‐lymphocyte precursors (CTLp) that lyse auto logo us tumor by a T‐cell receptor (TCR) dependent mechanism (specific CTLp) were evaluated by limiting dilution analysis (LDA) using lymphocytes from peripheral blood (PBL) and from surgically resected, tumor‐invaded lymph nodes (LNL) in 9 melanoma patients. The frequency of specific CTLp was determined in PBLs and/or LNIs of all patients by a modified LDA assay, enabling us to measure lytic activity on the autologous tumor that could be significantly inhibited by an anti‐CD3 monoclonal antibody (MAb). This assay allowed us to detect frequencies of specific CTLp ranging from 1/720 to 1/32,037 in peripheral blood and from 1/328 to 1/22,061 in tumor‐invaded lymph nodes. These frequencies indicated that lymphoid populations from PBLs or LNLs of melanoma patients may contain as low as 30 to as much as 3,000 specific CTLp/106 lymphocytes. In addition, comparison of wells containing specific CTLp with those showing no inhibition by anti‐CD3 MAb indicated that specific CTLp represent between 3 and 88% of all precursors with lytic activity on the tumor. In 6 of 9 patients, no marked differences between PBLs and LNIs in specific CTLp frequencies were found. A 10‐fold increase of specific CTLp, in comparison to PBL and LNL, was found only in lymphocytes isolated from a subcutaneous metastasis of one patient. Our results indicate that CTLp interacting with autologous tumor by a TCR‐dependent mechanism exist in PBL and LNL of most melanoma patients, although a wide variation in their absolute number is evident among different patients.
High levels of transglutaminase expression in doxorubicin‐resistant human breast carcinoma cells
Authors: Mehta Kapil
Published: February 2007

Tissue type II transglutaminase (TGase) is a member of the TGase family that catalyzes Ca2+‐dependent covalent cross‐linking of several amines to the γ‐carboxamide group of protein‐bound glutamine residues. The degree of therapeutic efficacy or toxicity of drugs may be related to their ability to serve as a substrate for TGase and their covalent linkage to glutamine residues of regulatory proteins through the catalytic action of this enzyme. Here, doxorubicin (adriamycin)‐resistant human breast carcinoma MCF‐7ADR cells exhibited 40‐ to 6C‐fold higher TGase activity than control drug‐sensitive MCF‐7wt cells. The same was observed in vivo: a small proportion of tumor cells became positive for TGase after administration of adriamycin‐based chemotherapy to patients with breast carcinoma. Similarly, continuous culture of MCF‐7WT, cells in the presence of adriamycin led to the appearance of the drug‐resistant phenotype that was in turn associated with increased expression of TGase. This increase in TGase was specific for adriamycin resistance. Like most known TGases, MCF‐7ADR TGase was completely dependent on the presence of Ca2+ for its catalytic activity. Based on its immunoreactivity, the TGase in MCF.7ADR cells was identified as an 85‐kDa tissue‐type TGase and was present only in the soluble form. Immunoblot analysis revealed that the increase in TGase activity was due to accumulation of the protein. Two cytosolic proteins of approximately 20 and 30 kDa in MCF‐7 cells served as suitable acyl donor substrates in TGase‐catalyzed reactions.
Therapy of human T‐cell acute lymphoblastic leukaemia in severe combined immunodeficient mice with two different anti‐CD7‐saporin immunotoxins containing hindered or non‐hindered disulphide cross‐linkers
Authors: Flavell David J. , Boehm Deborah A. , Okayama Keiko , Kohler Janice A. , Flavell Sopsamor N. U.
Published: February 2007

A SCID mouse model of human T‐ALL has been used to determine the in vivo therapeutic efficacy of two anti‐CD7‐saporin immunotoxins constructed with either a hindered (HB2‐SMPT‐Sap) or non‐hindered (HB2‐SPDP‐Sap) disulphide bond between antibody and saporin. Groups of 10 SCID mice were injected intravenously (i.v.) with 2 × 106 human T‐ALL HSB‐2 cells followed seven days later by i.v. injection with either a single dose or with 3 doses of HB2‐SPDP‐Sap or HB2‐SMPT‐Sap given on alternate days. Control groups received equivalent sham injections of PBS or molar equivalent amounts of unconjugated HB2 antibody + saporin. Animals receiving a single dose of HB2‐SMPT‐Sap showed better survival than animals receiving a single dose of HB2‐SPDP‐Sap but the difference was not shown to be significant by log‐rank analysis. When given as a triple dose both immunotoxins performed similarly. Comparison of single‐dose with triple‐dose IT therapy revealed that the therapeutic effect of a triple dose of HB2‐SPDP‐Sap was significantly better than that of single dose, but this was not the case with HB2‐SMPT‐Sap. Pharmacokinetic studies of HB2‐SPDP‐Sap and HB2‐SMPT‐Sap in normal and HSB‐2 leukaemia bearing SCID mice failed to reveal any difference in clearance rates for these two IT's. We conclude from these studies that there is no therapeutic advantage to be gained from constructing the HB2‐Sap IT with a hindered disulphide bond in this particular model of human T‐ALL.
Different characteristics of hepatoid and non‐hepatoid α‐fetoprotein‐producing gastric carcinomas: An experimental study using xenografted tumors
Authors: Aizawa Kikuo , Motoyama Teiichi , Suzuki Satoshi , Tanaka Norio , Yabusaki Hiroshi , Tanaka Shinsuke , Muto Ichiro , Tanaka Otsuo , Hatakeyama Katsuyoshi
Published: February 2007

The characteristics, including metastatic potential, of 5 xenografts of α‐fetoprotein (AFP)‐producing gastric carcinomas in nude mice, designated TSG1, TSG3, TSG11, TSG17 and TSG20, were examined. Of these xenografts, TSG1, TSG11 and TSG20 were regarded as hepatoid adenocarcinomas based on their morphological resemblance to hepatocellular carcinoma, frequent immunoreactivity for liver‐cell markers, and excessive production of AFP with a high concanavalin A (Con‐A)‐binding property of hepatic type. On the other hand, TSG3 and TSG17 tumors showed the features of poorly differentiated medullary adenocarcinoma with scattered AFP‐positive cells consistent with low AFP levels in mouse sera, and negative immunoreactivity for other liver‐cell markers. Ultrastructurally, these tumors were composed of undifferentiated cells with a little adenocarcinomatous differentiation. Moreover, the AFP produced by TSG3 and TSG17 tumors had an extremely high Con‐A non‐bound fraction (80% to 90%), which was different from that of the hepatic or yolk‐sac types. Therefore, both TSG3 and TSG17 tumors were regarded as non‐hepatoid, poorly differentiated adenocarcinomas which could be differentiated from any types of AFP‐producing gastric carcinoma. Furthermore, cells from hepatoid adenocarcinoma strains (TSG1, TSG11 and TSG20) injected into the spleens of nude mice produced liver metastases in all the mice examined, whereas cells from non‐hepatoid carcinoma strains (TSG3 and TSG17) produced few or no liver metastases. Our data show that some non‐hepatoid AFP‐producing gastric carcinomas have lower liver‐metastasizing potential than hepatoid AFP‐producing gastric carcinomas.
Hypermethylation of replicating hepatic DNA following N‐methyl‐N‐nitrosourea administration
Authors: Kanduc Darja , Aresta Antonella , Farber Emmanuel
Published: February 2007

Changes in the degree of methylation of cytosine in DNA are considered to be mechanistically important in modulating gene expression. To gain a better understanding of the relationship(s) linking onco‐proliferative processes and enzymatic DNA methylation, a study has been carried out on the hepatic DNA methylation pattern during DNA replication following partial hepatectomy (PH), mitogen treatment and N‐methyl‐N‐nitrosourea (MNU) administration in rats. The following results were obtained: (i) DNA hypomethylation was seen during DNA synthesis, with each of the 3 stimuli, namely MNU administration, partial hepatectomy, and hepatomitogen treatment; (ii) the level of DNA hypomethylation was not quantificatively related to the extent of DNA replication as measured by incorporation of [3H]thymidine into hepatic DNA: (iii) MNU administration under conditions conducive to carcinogenic development, i.e. during the S phase of compensatory cell proliferation, caused hypermethylation of replicating hepatic DNA, as shown by Hpall and Mspl restriction patterns.
Inhibition of the metastatic spread and growth of B16‐BL6 murine melanoma by a synthetic matrix metalloproteinase inhibitor
Authors: Chirvi Renato G. S. , Garofalo Angela , Crimmin Michael J. , Bawden Lindsay J. , Stoppacciaro Antonella , Brown Peter D. , Giavazzi Raffaella
Published: February 2007

The synthetic matrix metalloproteinase inhibitor batimastat was tested for its ability to inhibit growth and metastatic spread of the B16‐BL6 murine melanoma in syngeneic C57BL/6N mice. Intraperitoneal administration of batimastat resulted in a significant inhibition in the number of lung colonies produced by B16‐BL6 cells injected i.v. The effect of batimastat on spontaneous metastases was examined in mice inoculated in the hind footpad with B16‐BL6 melanoma. The primary tumor was removed surgically after 26‐28 days. Batimastat was administered twice a day from day 14 to day 28 (pre‐surgery) or from day 26 to day 44 (post‐surgery). With both protocols, the median number of lung metastases was not significantly affected, but there was a significant reduction in the weight of the metastases. Finally, the effect of batimastat was examined on s.c. growth of B16‐BL6 melanoma. Batimastat administered daily, starting at day of tumor transplantation, resulted in a significant growth delay, whereas treatment starting at advanced stage tumor only reduced tumor growth marginally. Our results indicate that a matrix metalloproteinase inhibitor can not only prevent the colonization of secondary organs by B16‐BL6 cells but also limit the growth of solid tumors.
Transformation and tumor progression are frequently associated with expression of the α3/β1 heterodimer in solid tumors
Authors: Bartolazzi Armando , Cerboni Cristina , Nicotra Maria R. , Mottolese Marcella , Bigotti Aldo , Natali Pier G.
Published: February 2007

The ability of tumor cells to interact with the extracellular matrix (ECM) is functionally mediated by a variety of receptor molecules among which the integrins are a well‐characterized family of mediators. In this study we have investigated immuno‐histochemically the in vivo expression of the α/β1 promiscuous receptor for ECM constituents in a variety of human solid malignancies. Although the receptor appears to undergo changes in distribution patterns, its expression is maintained in a high percentage of primary (76%) and metastatic (82%) tumors. Furthermore, the comparative immunohistochemical evaluation of α/β1 and its ligands, in a selected number of tumors of different histotypes, demonstrated that the expression of this integrin correlates with the presence of at least one ligand, either around nests of neoplastic cells or at the epithelial‐stromal interface. The highly conserved expression of α/β1 shown in this study suggests that this receptor may play a role in tumor growth at the primary as well as at the metastatic site.
Clonal analysis of human gynecologic cancers by means of the polymerase chain reaction
Authors: Sawada Masumi , Azuma Chihiro , Hashimoto Kazumasa , Noguchi Shinzaburo , Ozaki Masami , Saji Fumitaka , Tanizawa Osamu
Published: February 2007

Clonality of human gynecologic cancers was analyzed in small DNA samples prepared from cryostat sections, by means of the polymerase chain reaction (PCR). The method used for clonal analysis was based on restriction fragment length polymorphism of the X‐chromosome‐linked phosphoglycerokinase (PGK) gene and on the differential methylation of the PGK gene due to random inactivation of 1 of 2 X‐chromosomes by methylation in females. Among 52 gynecologic cancers tested, 25 were found to be heterozygous for the BstXI polymorphism of the PGK gene. All the 25 gynecologic cancers (4 cervix, 11 endometrium, 7 ovary and 3 fallopian tube) analyzed by the PCR‐based method were monoclonal in origin while adjacent normal tissues were polyclonal. When DNA samples were prepared from widely separated sites of tumors and/or metastatic lesions, every sample was found to be monoclonal, and the same allele of the PGK gene was inactivated in each case. These results demonstrate that clonal analysis by PCR offers a good method for studying clonality in small DNA samples prepared from cryostat sections of tumors. This method could be applied to distinguish between benign and malignant gynecologic lesions.
Polyclonal uses of T‐cell receptor (TCR)α and β genes for cytotoxic T lymphocytes in human metastatic melanoma: possible involvement of TCRα in tumor‐cell recognition
Authors: Seito Dai , Morita Tatsuo , Masuoka Kazuhiro , Maeda Tomoyuki , Saya Hideyuki , Itoh Kyogo
Published: February 2007

Identification of genetic structure and diversity of T‐cell receptor (TCR)α and β genes for cytotoxic T lymphocytes (CTLs) infiltrating human cancers is important for the better understanding of molecular mechanisms of host defense at tumor sites. cDNAs of TCRα and β genes of 22 different melanoma‐specific CTL clones established from the tumor‐infiltrating lymphocytes of 2 patients were sequenced for analysis of their genetic structure and diversity. Vα7.2‐Jα10‐Cα was found in 4 of 22 clones, 2 of which also used the same β‐chain. The other 20 clones showed different combinations of α and β use. At deduced amino‐acid levels, 7 of 9 clones from one patient used a threonine residue at the 26th position in the complementarity‐determining region (CDR)1 of TCRα. Eight of 13 clones used a threonine at the 99th or a serine residue at the 100th position in CDR3 of TCRα. CTL clones with the same or different TCRα showed the same or different patterns of cytotoxicity, respectively. These results suggest that CTLs usually do not demonstrate clonal expansion at tumor sites of metastatic melanoma's but rather that polyclonal T cells capable of binding to multiple melanoma determinants through CDR3 of TCRα accumulate in the tumor.
A K‐ras 13GLY → ASP mutation is recognized by HLA‐DQ7 restricted T cells in a patient with colorectal cancer. Modifying effect of DQ7 on established cancers harbouring this mutation?
Authors: Fossum Beate , Breivik Jarle , Meling Gunn Iren , Iii Tobias Gedde‐Dahl , Hansen Torbjørn , Knutsen Ingebjørg , Rognum Torleiv O. , Thorsby Erik , Gaudernack Gustav
Published: February 2007

We have characterized and described in detail 2 CD4+ T‐lymphocyte clones (TLC) from a colonic cancer patient. These TLC specifically recognize a K‐ras‐derived peptide carrying the 13Asp mutation commonly found in adenocarcinomas of the colon. The TLC were independently derived, as they carried 2 different T‐cell receptors. The TLC recognized partly overlapping epitopes within the 13Asp peptide, presented by HLA‐DQ7 molecules, suggesting that this molecule might confer some protective immunity against the mutation. On the basis of analysis of 251 colonic carcinomas, the presence of HLA‐DQ7 did not seem to protect against the establishment of carcinomas carrying the 13Asp mutation, since the frequency of the DQ7 haplotype was not decreased among patients having this mutation. A modifying effect of DQ7 on the development of carcinomas with a 13Asp mutation was, however, observed, resulting in fewer tumours reaching advanced Dukes stages when DQ7 was present.
Pyrimidine nucleotide metabolism in human colon carcinomas: Comparison of normal tissues, primary tumors and xenografts
Authors: Luccioni C. , Beaumatin J. , Bardot V. , Lefrançois D.
Published: February 2007

The activities of 5 enzymes involved in the pyrimidine metabolism were measured in xenografts of 8 human colon adenocarcinomas and in the corresponding primary tumors and normal tissues. The enzymes studied were thymidine kinase, thymidine phosphorylase, uridine kinase, uridine phosphorylase and thymidylate synthase. With the exception of the phosphorylases in one tumor, all enzyme activities were higher in primary tumors than in the corresponding normal tissues. The average activities of thymidine kinase and thymidylate synthase were of the same order of magnitude in xenografts and in primary tumors. The uridine metabolizing enzymes tend to have a higher activity in xenografts than in primary tumors. The most consistent and significant change was a sharp decrease in thymidine phosphorylase activity in xenografts as compared to primary tumors. Whether or not the difference in thymidine phosphorylase activity between xenografts and primary tumors is related to the contribution of non‐cancerous cells in primary tumors remains to be determined. However, these results raise questions concerning the representativeness of xenografts with reference to primary tumors and suggest that care should be taken in the application of this model.
Interferon gamma‐induced reduction in erbB‐2 tyrosyl phosphorylation in human ovarian carcinoma cells
Authors: Mishra Shirkant , Hamburger Anne W.
Published: February 2007

Interferon, which inhibits growth of ovarian cancer cells in vivo and in vitro, decreases expression of erbB‐2 protein in ovarian carcinoma cell lines. We now show that interferon‐γ (IFN‐γ) also decreases constitutive tyrosine phosphorylation of erbB‐2 and inhibits erbB‐2 kinase activity in an ovarian cancer cell line. SK‐OV3 ovarian cancer cells, which over‐express erbB‐2, were treated with IFN‐γ for 0‐72 hr. Immunoblot analysis revealed that IFN decreased the levels of tyrosyl phosphorylated erbB‐2 24 hr after IFN treatment. Protein levels of erbB‐2 were not changed until 72 hr post‐treatment. Tyrosine kinase (TK) activity of immunoprecipitated erbB‐2 for an exogenous substrate was decreased in IFN‐treated cells. Total cellular protein tyrosine phosphatase (PTPase) activity for the epidermal growth factor receptor was not changed by IFN treatment. Our results suggest that the decreased levels of tyrosyl phosphorylated proteins observed after IFN treatment in SK‐OV3 cells may be due to inhibition of erbB‐2 kinase activity.
Suppression of multi‐drug resistance gene expression in the mouse liver by 1,4‐bis[2,(3,5‐dichloropyridyloxy)]benzene
Authors: Russell Alison L. , Henderson Colin J. , Smith Gillian , Roland Wolf C.
Published: February 2007

P‐glycoproteins encoded by the (multi‐drug resistance) mdr genes play a central role in the resistance of tumor cells to a wide range of anti‐cancer drugs. Modulation of P‐glycoprotein function could therefore provide a means of sensitising tumor cells to chemotherapy. Studies in this context have centred around the use of compounds which antagonise the P‐glycoprotein membrane transport system. To investigate the possibility of modulating P‐glycoprotein expression at a transcriptional level, we investigated the effects of hormonal factors and cytochrome P450‐inducing agents on hepatic expression of murine mdr I, mdr 2 and mdr 3. Hepatic mdr 2 and mdr 3 expressions were significantly suppressed in hypophysectomised animals, indicating that pituitary hormones activate the hepatic expression of these genes. Many of the foreign compounds and anti‐cancer drugs tested did not significantly induce mdr 1, 2 or 3 expression. However, it was of particular interest that a potent cytochrome P450 inducer, 1,4‐bis[2‐(3,5‐dichloropyridyloxy)]benzene, almost completely suppressed hepatic mdr 2 and 3 expressions.
Vaccination with ant‐idiotype antibodies mimicking a renal cell carcinoma‐associated antigen induces tumor immunity
Authors: Uemura Hirotsugu , Beniers Antonius J. M. C. , Okajima Eigoro , Debruyne Frans M. J. , Oosterwijk Egbert
Published: February 2007

We have previously isolated and characterized 6 different internal image mouse monoclonal anti‐idiotype antibodies (Ab2) directed against the paratope of mouse monoclonal antibody G250 (MAbG250, Abl), which specifically reacts with human renal cell carcinoma (RCC). These Ab2s (NUH31,44,51, 71,82 and 91) demonstrated specificity for the combining site of AbI, and appeared to recognize 2 partly overlapping idiotopes on AbI. In this study, we further characterize the fine specificity of the Ab2, investigate whether the immunogenicity of Ab2 could be enhanced by conjugation to a carrier and investigate the anti‐tumor efficacy of Ab3 sera in mice challenged with RCC. Immunization of animals with Ab2 conjugated to keyhole limpet hemocyanin as carrier protein resulted in a 2‐fold increase in antigen‐specific anti‐anti‐idiotype antibodies (Ab3) as compared with immunization using Ab2 alone. Specific reactivity was observed with antigen‐positive cell lysates, and all Ab3 sera contained immunoglobulin resembling AbI (Abl), as shown by competitive AbI ‐antigen binding assays. Fine‐specificity studies of Ab3 sera revealed that the Ab2s can be divided into 4 mutually exclusive groups, showing that the 6 Ab2s recognize 4 slightly different idiotopes in the Abl binding pocket. Treatment of RCC‐challenged mice with Ab3 sera resulted in significant tumor growth inhibition and lower tumor take rates as compared with control groups. Ab3 sera obtained from NUH‐91‐immunized animals showed superior characteristics as compared to the other Ab3 sera: no tumors remained after 5 weeks of Ab3‐NUH91 treatment. Our findings indicate that the Ab2 elicit powerful anti‐tumor effects in immune‐competent animals.