bluuebunny/crossref_metadata_2025 huggingface dataset:
Biased utilization of immunoglobulin variable region heavy‐ and light‐chain genes by the malignant CD5‐ B lymphocytes from patients with Burkitt's lymphoma
Authors: Moazzeni Mohammad , Mosayyebi Ghasem , Stevenson Freda K. , Abbot Sandra , Mageed Rizgar A. , Shokri Fazel
Published: February 2007

Twenty‐six established Burkitt's lymphoma (BL) cell lines from endemic or sporadic groups of patients were examined for the expression of cross‐reactive idiotypes (CRI) associated with VHI, VHIII, VHIV, VHVI, VKIIIa and VKIIIb heavy‐and light‐chain gene products, using a panel of anti‐CRI and anti‐subgroup monoclonal antibodies (MAbs). Membrane, cytoplasmic and secreted immunoglobulins (Ig) were analysed by immunofluorescence, immunoperoxidase and ELISA respectively. While 35% of the lines expressed either of the VHIV‐associated CRI, recognised by the MAbs 9G4 or LCI, none expressed the other VH‐associated CRI included in our study. Of the kappa light chain expressing BL lines 54% and 46% belonged to the VKIII subgroup and VKIIIb sub‐subgroups respectively. None, however, was found to express the VKIIIa or VKIIIb‐associated CRI, recognised by the 6B6.6 and 17‐109 MAbs. A significant association has been observed between the expression of the VHIV‐associated CRI and the VKIII subgroup within the BL lines derived from the sporadic group of patients as compared with their endemic counterparts. Our results suggest that the expressed repertoire of Ig variable region genes within the malignant B lymphocytes of BL is not random and that a highly selective mechanism(s) may operate on this subset of B lymphocytes, as evidenced by the expression of the VH and VK gene products.
Increased UV‐induced SCEs but normal repair of DNA damage in p53‐deficient mouse cells
Authors: Ishizaki Kanji , Ejima Yosuke , Matsunaga Tsukasa , Hara Ryujiro , Sakamoto Ayako , Ikenaga Mituo , Ikawa Yoji , Aizawa Shin‐Ichi
Published: February 2007

UV‐induced sister chromatid exchanges (SCEs) in p53‐deficient mouse cells were studied to obtain more evidence regarding the involvement of p53 protein in the DNA repair pathway as a checkpoint protein. After 5 J/m2 UV irradiation, mutant‐type homozygous cells for p53‐deficiency showed the same number of SCEs as the heterozygous and wild‐type homozygous cells. In the heterozygous and wild‐type homozygous cells, no further increase of SCEs was observed after 10 J/m2 UV irradiation. In contrast, in mutant‐type homozygous cells about twice as many SCEs were induced by 10 J/m2 UV as by 5 J/m2 UV. In mutant‐type homozygous cells, fractions of S‐phase cells decreased just after 10 J/m2 UV irradiation, but recovered to higher than control levels within a short time, while in heterozygous and wild‐type homozygous cells, the decrease in S‐phase cells was prolonged by more than 6 hr and no increase above control levels was observed. Although no difference in UV sensitivity and repair of UV‐induced DNA damage was found among the 3 genotypes, which were determined by the relative colony‐forming ability after UV irradiation and removal of thymine dimers and (6‐4) photoproducts from cellular DNA, our data strongly suggest an impaired checkpoint function in p53‐deficient cells when DNA is damaged.
Development of vincristine resistance and increased sensitivity to cyclosporin A and verapamil in the human U‐937 lymphoma cell line without overexpression of the 170‐KDa P‐glycoprotein
Authors: Botling Johan , Liminga Gunnar , Larsson Rolf , Nygren Peter , Nilsson Kenneth
Published: February 2007

A vincristine (Vcr)‐resistant subline of the human histiocytic lymphoma cell line U‐937 (U‐937‐vcr) has been established and characterized with respect to its phenotypic features, including growth rate, surface marker expression and ability to respond to differentiation‐inducing agents. The sensitivity of U‐937‐vcr cells to the direct cytotoxicity of cyclosporin A (CsA) and verapamil (Ver), and the capacity of these drugs to modify Vcr resistance, were also examined. The U‐937‐vcr cells exhibited a more than 200‐fold resistance to Vcr, and cross‐resistance to vinorelbin and taxol. Also, there was a slight cross‐resistance to colchin, doxorubicin and VP16. However, the response of U‐937‐vcr to CsA or Ver alone was substantially altered, with a marked decrease in their respective IC50s. The U‐937‐vcr cells did not show increased levels of pgp 170. We conclude that the development of Vcr resistance was not associated with a change in the major phenotyic properties of the U‐937 cell line, and that resistance modifier hypersensitivity was not associated with increase in pgp 170 expression.
Scattered micrometastases visualized at the single‐cell level: Detection and re‐isolation of lacZ‐labeled metastasized lymphoma cells
Authors: Krüger Achim , Schirrmacher Volker , Hoegen Paul Von
Published: February 2007

To study metastasis at the single‐cell level we transduced highly metastatic ESb lymphoma cells with a retroviral expression vector containing the lacZ (bacterial β‐galactosidase) gene. This allowed single ESb‐lacZ tumor cells to be detected in infiltrated target organs by means of X‐Gal staining. Despite expression of the lacZ gene, the tumor cells were still tumorigenic, highly metastatic, unchanged in phenotype and therefore comparable to parental ESb cells. After spontaneous metastasis, whole‐organ staining revealed metastatic foci at the surface of the liver. In histological liver sections, metastatic clusters and single dispersed tumor cells could be detected. In contrast to whole‐organ staining, histological examination revealed scattered distribution of tumor cells throughout the organ, which was not evident with parental ESb cells. In addition, clusters with diffuse or dense (focal) appearance were found, in correlation with the whole‐organ staining. Expression of the foreign lacZ gene allowed the metastatic spread of tumor cells to liver and spleen to be quantified approximately by FACS analysis. Furthermore, it was shown that the newly expressed β‐gal was expressed not only intercellularly but also at the cell surface. There it could be recognized by MAbs and cytotoxic T‐cells (CTL). β‐gal did not affect CTL recognition of the ESb tumor‐associated antigen. In conclusion, lacZ could be used as a genetic marker for a highly metastatic lymphoma, to define scattered metastatic spread in the liver at the single‐cell level and to quantify the tumor load by FACS analysis.
Tumorigenicity of BALB3T3 A31 cells transfected with hamster‐complement‐C1s cDNA
Authors: Sakai Norie , Kusunoki Mayumi , Nishida Munehiro , Toyoguchi Toru , Fukutomi Hisayuki , Sakiyama Hisako
Published: February 2007

Hamster‐complement‐CIs cDNA was inserted into an expression plasmid BCMGSNeo (BCMGSNeoHACS). BALB/c mouse fibroblast A31 cells, which do not produce CIs, were transfected with BCMGSNeoHACS and the transfectants were selected with G418. Normal CIs production by the transfectants was confirmed by Northern and immunoblot analysis and by an esterase assay. To examine the tumorigenicity of the transfectants, 1 × 106 cells were injected s.c. into 6‐week‐old BALB/c nu/nu mice. Three CIs cDNA transfectants (A3CS9, A3CS12, A3CS13) formed tumors whereas both A31 and A31 transfected with the vector alone (A3BCMI and A3BCM3) did not. The tumors derived from the transfectants showed invasive growth, and many capillaries were observed in the tumors. A tumor derived from A3CS13 was examined immunohistochemically and found to be reactive with an anti‐CIs monoclonal antibody. Tumor cells were cultured in vitro again and CIs secreted into the culture medium was examined by immunoblot analysis. CIs synthesized by the tumor cells derived from A3CS13 maintained its biological functions. Tumor cells derived from A3CS9 and A3CS12 cells, however, produced CIs having abnormal disulfide bonds.
Age‐period‐cohort modelling of large‐bowel‐cancer incidence by anatomic sub‐site and sex in Denmark
Authors: Dubrow Robert , Johansen Christoffer , Skov Torsten , Holford Theodore R.
Published: February 2007

In a previous investigation, statistical modelling was used to examine the relationship between large‐bowel‐cancer incidence and age, time period and birth cohort by anatomic sub‐site and sex, using data from the Connecticut Tumor Registry (CTR) for the period 1950 to 1984. This analysis revealed differences in age‐period‐cohort patterns that suggested etiologic distinctions among sub‐site groupings and between the sexes. To test the generalizability of the Connecticut findings, we have conducted a similar age‐period‐cohort analysis using data from the Danish Cancer Registry (DCR) for the period 1953 to 1987. Cancers of the large bowel were classified into 6 anatomic sub‐sites: cecum, ascending colon, transverse colon, descending colon, sigmoid colon and rectum. Data were fitted to log‐linear age‐period‐cohort models. If we interpret differences in age‐period‐cohort patterns as reflecting etiologic distinctions, the Denmark analysis, in conjunction with the Connecticut findings, was consistent with there being etiologic distinctions between cancers of the colon vs. the rectum in both males and females, between cancers of the cecum and the ascending colon vs. the remainder of the colon in females and between males vs. females for cancers of the sigmoid colon and rectum. Cancers of the cecum and the ascending colon were the most similar between males and females. Due to the ambiguities of age‐period‐cohort modelling, these should be considered only tentative conclusions that can be tested by analytical epidemiologic studies.
A member of the melanoma antigen‐encoding gene (MAGE) family is expressed in human skin during wound healing
Authors: Becker Jürgen C. , Gillitzer Reinhard , Bröcker Eva‐Bettina
Published: February 2007

MAGE‐1 has been identified as a human gene, which directs the expression of an antigen being recognized on melanoma cells by autologous cytolytic T cells. MAGE‐1 is expressed in melanomas and some other tumors. It has been proposed that this gene may be linked to the transformation event and therefore might serve as an approach to precisely targeted immunotherapy. Prior to such an approach, extensive testing of normal human tissue is necessary to establish the tumor‐specific nature of MAGE‐1 expression. Similar to events that occur during neoplastic tumor growth and spreading, wound healing involves a complex interrelationship between various cell types which migrate, proliferate and differentiate. Therefore, we investigated the expression of MAGE‐1 in skin during wound repair. We could detect MAGE‐1 mRNA by RT‐PCR followed by specific hybridization as well as by Northern blotting in human skin from the 1st to the 7th day after wounding. Comparison of the expression of MAGE mRNA with that of β‐actin mRNA showed that it is expressed in amounts equal to about and at least one‐fifth that of β‐actin. Our data strongly suggest that MAGE mRNA expression is not necessarily linked to neoplastic transformation, but rather represents the function of a cellular gene which is activated during inflammation or early tissue repair.
A search for unknown blood‐borne oncogenic viruses
Authors: Memon Anjum , Doll Sir Richard
Published: February 2007

In previous decades, infants who received blood transfusions shortly after birth or in utero might have been infected at a particularly vulnerable age by some blood‐borne oncogen it virus. A cohort of such infants has therefore been followed into adult life to see if they suffered any excess of neoplastic disease or of non‐neoplastic mortality.

A total of 12,690 infants were identified who were transfused between 1 January 1942 and 31 December 1970, in most cases for the prevention or treatment of haemolytic disease of the newborn. All but 361 (2.8%) were found to have been registered with a National Health Service (NHS) practitioner and were followed in the NHS central records until they died, emigrated, were removed from NHS lists or until 1 January 1992, whichever occurred first. Mortality and cancer incidence were compared with that expected from national rates. No marked disparity was observed and there was no excess of childhood leukaemia. The incidence of non‐Hodgkin's lymphoma at 15 to 49 years of age was about twice that expected, but the excess was not statistically significant.
Endogenous tumor necrosis factor functions as a resistant factor against adriamycin
Authors: Maeda Masahiro , Watanabe Naoki , Okamoto Tetsuro , Yoshiro Niitsu Naoki Tsuji
Published: February 2007

One of the mechanisms of cytotoxicity by tumor necrosis factor (TNF) and heat is the induction of reactive oxygen molecules. Cells producing endogenous tumor necrosis factor (enTNF) show resistance to the cytotoxicity of exogenous TNF and heat by inducing manganous superoxide dismutase (MnSOD) to scavenge the reactive oxygen molecules. Intracellular hydroxyl radical production is also involved in adriamycin‐induced cytotoxicity. In this study, we therefore examined the possibility that enTNF may act as a protective protein against adriamycin‐induced cytotoxicity in a manner similar to that in which it protects against exogenous TNF and heat. Adriamycin‐sensitive L‐M (mouse tumorigenic fibroblast) cells, originally expressing no enTNF, were transfected with an expression vector which directs the synthesis of non‐secretory‐type human TNF (enTNF). The stable transformants became resistant to adriamycin with increased levels of MnSOD. Conversely, when HeLa (human uterine cervical cancer) cells, which originally produce an appreciable amount of enTNF, were transfected with an anti‐sense TNF mRNA expression vector to inhibit enTNF synthesis, their intracellular MnSOD activity was suppressed and adriamycin sensitivity was enhanced. However, no alterations in expression of multidrug‐resistant gene products—P‐170 glycoprotein, glutathione S‐transferase π (GST‐π) and the intracellular concentrations of glutathione (GSH)‐were observed in these transfectants as compared to their parent cells. These results indicate that enTNF exerts its intracellular protective effect against adriamycin‐induced cytotoxicity by the same mechanism as that against exogenous TNF and heat, namely scavenging reactive oxygen with induced MnSOD.
Human recombinant interferons‐beta and ‐gamma decrease gelatinase production and invasion by human KG‐2 renal‐carcinoma cells
Authors: Gohji Kazuo , Fidler Isaiah J. , Tsan Rachel , Radinsky Robert , Von Eschenbach Andrew C. , Tsuruo Takashi , Nakajima Motowo
Published: February 2007

We investigated the effects of human recombinant interferons (r‐IFNs) on gelatinase production and invasion by human renal‐cell carcinoma (HRCC). Incubation of KG‐2 HRCC with human r‐IFN‐β or ‐γ (but not ‐α) suppressed transcription of the 72‐kDa gelatinase gene and, hence, production of gelatinase activity. These inhibitory effects of interferons (IFNs) were independent of their antiproliferative effects. Treatment of KG‐2 cells with r‐IFN‐β or ‐γ significantly inhibited cell invasion through reconstituted basement membrane toward chemoattractants produced by kidney fibroblasts. The inhibitory activity of r‐IFNs was specific to the KG‐2 cells since gelatinase activity by various fibroblasts was unaffected. These findings suggest that r‐IFN‐β or IFN‐γ may be used to inhibit the invasive potential of HRCC.
Hibernomas are characterized by rearrangements of chromosome bands 11q13‐21
Authors: Mertens Fredrik , Rydholm Anders , Brosjö Otte , Willéd Helena , Mitelman Felix , Mandahl Nils
Published: February 2007

Cytogenetic analysis revealed structural rearrangements of 11q13‐21 in 9 lipogenic tumors—4 hibernomas, 4 typical lipomas and I mixed myxoid and well‐differentiated liposarcoma. Two hibernomas and all lipomas simultaneously showed aberrations attributable to previously recognized cytogenetic subgroups among benign adipose tissue tumors, i.e., supernumerary ring chromosomes or rearrangements of 6p, 12q13‐15, or 13q. The sole anomaly in the liposarcoma was a 4‐way translocation t(9;11;22;12)(q21;q13;q11;q13), an aberration that has not previously been detected in either myxoid or well‐differentiated liposarcomas. Together with our 4 cases, a total of 7 cytogenetically aberrant hibernomas have been reported. Five tumors have shown rearrangements of 11q13 and 2 of 11q21, indicating that this segment of chromosome 11 harbors one or more genes of importance in hibernoma development. Rearrangements of 11q13‐21 also appear to be frequent in typical lipomas; a total of 8 typical lipomas with 11q13‐21 changes have been described, including those in our series. In all but I, however, breakpoints were also found in 6p, 12q13‐15, or 13q.
Widowhood and divorce related to cancer risk in middle‐aged women. A nested case‐control study among norwegian women born between 1935 and 1954
Authors: Kvikstad Anne , Vatten Lars J. , Tretli Steinar , Kvinnsland Stener
Published: February 2007

We have examined whether the risk of cancer among divorced or widowed Norwegian women born between 1935 and 1954 was any different from that of married women. Among a total of approximately 600,000 women, we applied a nested case‐control design. Thus, the study was population‐based and included 17,235 incident cases of cancer with 34,460 agematched controls. For widowed women, there was no overall relation with cancer. For divorced women, 2 strikingly different associations were apparent. A reduced risk was seen for cancers of a number of sites, including thyroid, endometrium, colorectum, and breast, as well as malignant melanoma and hematologic malignancies, with statistically significant estimates of relative risk ranging from 0.64 to 0.84. In contrast, divorced women had a strongly elevated risk of lung and cervical cancer. Moreover, there was a gradual reduction in the relative risk of cancer at some sites with increasing age at divorce, and with duration of marriage prior to divorce. In this study of middleaged women, the risk of cancer among widows was no different from that of married women. Divorced women had an increased risk of cancers which are related to cigarette smoking but, simultaneously, a reduced risk of cancer at a number of other sites. Since the negative associations for some cancers were strongly related to increasing age at divorce and to duration of marriage, the results may indicate that the reduction in risk is related to factors which characterize the marital period preceding divorce.
Association studies of a polymorphism in the Wilms' tumor 1 locus in norwegian patients with testicular cancer
Authors: Heimdal Ketil R. , Lothe Ragnhild A. , Fosså Sophie Dorothea , Børresen Anne‐Lise
Published: February 2007

One strategy for identifying genes involved in genetic predisposition to testicular germ‐cell tumors (TC) is to perform association studies with polymorphic loci at or closely linked to candidate genes. Genes involved in normal fetal genital development, such as the Wilms' tumor 1 gene (WT1) located at 11p13, are among such candidates. The present study compares a TC (n = 442) and a control (n = 384) population for the allele frequencies of 2 polymorphic loci located at chromosome band 11p13. One of the polymorphisms (WT) was located within and the other (D11S325) in close proximity to theWT1gene. No differences in allele frequencies between cancer patients and controls were found. However, the frequency of the A1 allele of the WT polymorphism was significantly increased in one of the cancer subgroups most likely to carry susceptibility genes (patients with bilateral cancer) compared to controls. Furthermore, the frequency of the A1 allele was increased in patients with metastatic disease. Such differences in allele frequencies were not observed for the D11S325 locus. The findings might indicate an involvement of theWT1gene both in susceptibility to TC and in progression of the disease.
Apoptotic cell death induced by a mouse‐human anti‐APO‐1 chimeric antibody leads to tumor regression
Authors: Coney Leslie R. , Daniel Peter T. , Sanborn David , Dhein Jens , Debatin Klaus‐Michael , Krammer Peter H. , Zurawski V. R.
Published: February 2007

The murine anti‐APO‐1 antibody (γ3, k) induces programmed cell death (apoptosis) following binding to the APO‐I antigen (m.w., 48 kDa) expressed, e.g., on activated or malignant lymphocytes. APO‐I expression on malignant cell lines and tissues suggested potential clinical utility supported by anti‐APO‐I‐mediated tumor regression in a nude mouse model. A mouse‐human anti‐APO‐1 chimeric antibody (γ3, k) with an affinity similar to that of the murine antibody was produced. Chimeric anti‐APO‐1 showed the same potential to inhibit growth of the SKW6.4 B‐lymphoblastoid cell line as murine anti‐APO‐1, In addition, both the chimeric and murine anti‐APO‐1 antibodies were equally capable of mediating complete macroscopic tumor regression of a SKW6.4 xenotransplant in SCID mice by induction of apoptosis. Induction of apoptosis was the only mechanism for tumor regression because neither murine nor chimeric anti‐APO‐1 showed anti‐tumor activity against solid H53 tumor (APO‐1 antigen‐positive, anti‐APO‐1 ‐resistant) xenotransplants. Our results indicate that the chimeric anti‐APO‐1 antibody effectively induces apoptosis and suggest that chimeric anti‐APO‐1 should be evaluated for the treatment of malignant cells expressing the APO‐1 antigen. However, chimeric anti‐APO‐1 might only be used therapeutically when the antibody can be targeted specifically to tumor cells.
Cloning of feline p53 tumor‐suppressor gene and its aberration in hematopoietic tumors
Authors: Okuda Masaru , Umeda Akiko , Sakai Tadashi , Ohashi Takashi , Momoi Yasuyuki , Youn Hwa‐Young , Watari Toshihiro , Goitsuka Ryo , Tsujtmoto Hajimc , Hasegawa Atsuhiko
Published: February 2007

Alterations of the p53 tumor‐suppressor gene have been observed in a variety of human and mouse tumors. For investigation of the role of this gene in tumors of cats, feline p53 cDNA was molecularly cloned from a feline lymph‐node cDNA library. The cloned cDNA (FF53) contained the whole open reading frame of p53 gene encoding 386 amino acids. The amino‐acid sequence of the feline p53 gene showed 82.1% and 74.9% similarities with those of the human and mouse counterparts, respectively, and had structural characteristics in common with the p53 genes of several other species. Aberrations of the p53 gene were investigated by RT‐PCR and single‐strand conformation polymorphism analyses. Of 10 primary hematopoietic tumors and 3 lymphoma cell lines examined, one lymphoma and one lymphoma cell line had a point mutation of the p53 gene, resulting in single amino‐acid substitutions.
Correlation between stromelysin‐3 mRNA level and outcome of human breast cancer
Authors: Engel Georg , Heselmeyer Kerstin , Auer Gert , Bäckdahl Martin , Eriksson Elina , Under Stig
Published: February 2007

The expression level of strometysin‐3 (ST3) mRNA was analyzed by in situ hybridization of formalin‐fixed, paraffin‐embedded primary breast‐tumor samples from 76 patients. Digital image analysis of the dark‐field in situ hybridization signal was used to measure the maximal level of ST3 expression in each tumor. All 55 invasive ductal carcinomas and 9 of 10 invasive lobular carcinomas were positive for ST3. Invasive tumors had significantly higher levels of ST3 than in situ tumors. Furthermore, ST3 levels were higher in invasive ductal carcinomas than in invasive lobular carcinomas. The ST3 expression level was significantly correlated to fatal metastatic disease (mean follow‐up 104 months). ST3 levels of < 2,500 units were associated with distant metastasis in 46% of patients, whereas levels of >2,500 units were associated with metastasis in 79% of patients selected for study. ST3 mRNA levels did not correlate with tumor siie, microvessel density, DNA pioidy or estrogen‐receptor levels. Studies of ST3 expression may provide information valuable for the understanding of breast cancer biology and for prognosis.
Expression of an endogenous retroviral gene product in human placenta
Authors: Kitamura Masanori , Maruyama Naoki , Shirasawa Takuji , Nagasawa Ryuji , Watanabe Kazutada , Tateno Masatoshi , Yoshiki Takashi
Published: February 2007

To investigate the presence and potential pathophysiological role of endogenous retroviruses in humans, we prepared a recombinant protein using clone 4‐1, a proviral sequence. DNA fragments containing the env region of clone 4‐1 were subdoned into a prokaryotic expression vector (pET3), and 2 fusion proteins, SU413 and SU415, were then expressed in Escherichia coli after treatment with isopropyl‐β‐thiogalactopyranoside (1PTG). By sonicating lysates of the transformed E. coli, the recombinant protein SU4I3 was successfully separated from the native bacterial components, and was used to raise an antiserum in rabbits. In immunoblot analysis, this antiserum specifically recognized the recombinant protein, but did not react with other components of E. coli. This antiserum was then used for an immunofluorescence study of human placenta, in which the env gene transcript has been reported. As a result, the anti‐SU4l3 serum detected substances in syncytiotropho‐blasts and vascular endothelia from a human placenta. No such reactivity was detected in human kidney or human liver. Immunoblot analysis revealed that this antiserum reacted to a single molecule of 38‐kDa in placenta, and its reactivity was reduced by the antiserum absorbed with SU4I3 antigen. These findings suggest that human placental syncytiotrophoblasts and vascular endothelia preferentially express a molecule encoded by human endogenous retrovirus clone 4‐1.
Collagens, integrins and the mesenchymal drift in glioblastomas: A comparison of biopsy specimens, spheroid and early monolayer cultures
Authors: Paulus Werner , Huettner Claudia , Tonn Jorg C.
Published: February 2007

To analyze the process of mesenchymal differentiation in vitro, we examined 5 human glioblastomas as biopsy specimens, monolayer cultures and 3‐dimensional fragment spheroid cultures for the immunohistochemical expression of extracellular matrix (ECM) components (collagen types I, III ‐VI, laminin) and integrin receptors (β1, β2, β3 and β4 chains). mRNA for type‐I and type‐IV collagen at chains was quantified using reverse transcription‐polymerase chain reaction. In situ, glioma cells expressed β1, the common β chain of most integrin ECM receptors, while ECM components were restricted to vascular elements. Early monolayer cultures showed a marked increase in ECM components (interstitial collagen? more than basement membrane components), and coexpression of ECM components and glial fibrillary acidic protein (GFAP) by most cells, β2 and β3 integrins were upregulated in the primary cultures. In the fifth passages, GFAP‐positive cells were decreased and collagen‐expressing cells increased. The spheroids exhibited preserved GFAP staining, neoexpression of β4 integrin in some tumors, and variable ECM expression by glioma cells which was lower than that in monolayer cultures. ECM deposition usually commenced in central spheroid areas where the Ki‐67 proliferation index was low. We conclude that different culture systems are characterized by distinct expression patterns for ECM components and receptors, and that mesenchymal features in cultured gliomas arise due to transdifferentiation of glioma cells.
Possible involvement of multidrug‐resistance‐associated protein (MRP) gene expression in spontaneous drug resistance to vincristine, etoposide and adriamycin in human glioma cells
Authors: Hasegawa Shuji , Taniguchi Ken , Yokomizo Akira , Kuwano Takashi , Ono Mayumi , Mori Temaki , Hori Shigeaki , Kohno Kimitoshi , Kuwano Michihiko
Published: February 2007

The multidrug‐resistance phenotype in human tumors is partly associated with over‐expression of the 170 kDa‐P‐glycoprotein encoded by the multidrug‐resistance‐1 (MDRI) gene. Another related, but non‐P‐glycoprotein, multidrug‐resistance‐associated protein (MRP) gene encodes a 190 kDa membrane ATP‐binding protein. Glioblastoma multiforme is a highly malignant primary neoplasm of the central nervous system which is refractory to anti‐cancer chemotherapy, but the mechanism underlying this drug resistance is unknown. Out of glioma cell lines, 2, namely IN500 and T98G, which had elevated MRP mRNA levels, showed the highest resistance to multiple anti‐cancer agents such as etoposide, vincristine and adriamycin, and decreased intracellular accumulation of etoposide. In the remaining 5 cell lines, various degrees of sensitivity to adriamycin and etoposide appeared to correlate with their respective MRP mRNA levels. Our study proposes that MRP may be involved in spontaneous multidrug resistance in human gliomas.
S‐phase fraction after gating on epithelial cells predicts recurrence in node‐negative breast cancer
Authors: Wingren Sten , Stå Olle , Sullivan Siw , Brisfors Ann , Nordenskjöld Bo
Published: February 2007

We have compared the prediction of distant recurrence for S‐phase fraction (SPF) and DNA‐ploidy, as estimated by flow cytometry, on an epithelial cell population and an unselected cell population from 268 node‐negative breast‐cancer patients diagnosed between 1985 and 1988. The tumor tissue was mechanically disintegrated and divided for flow cytometric analysis using both gated cells containing cytokeratin 8 and 18 and ungated cells treated with a detergent‐trypsin solution. The relationship to distant recurrence was investigated for flow cytometric data, tumor size and estrogen and progesterone receptor content in univariate and multivariate Cox's regression analysis. The regression analyses were performed on 209 cases with S‐phase fractions estimated by both methods. In 11 cases, DNA‐ploidy classification differed, reflecting increased sensitivity to minor aneuploid peaks but a decreased ability to separate peaks in the near‐diploid region for the gated populations. When SPF were used in univariate analysis as a continuous parameter or the upper tertile was used as cut‐off value, SPF from the cytokeratin‐gated cell population were most closely associated with recurrence and contributed additional prognostic information to SPF from the unselected cell population in the multivariate analysis. Out of the following variables:tumor size, ER and PR status, SPF and DNA ploidy, only SPF from immunoselected cells contributed prognostic information in multivariate analysis. These results indicate that SPF from immunoselected cell populations improves the prediction of recurrence in node‐negative breast cancer.
Distribution of oncofetal fibronectin isoforms in normal, hyperplastic and neoplastic human breast tissues
Authors: Kaczmarek Janusz , Castellani Patrizia , Nicolo Guido , Spina Bruno , Allemanni Giorgio , Zardi Luciano
Published: February 2007

Two different oncofetal fibronectins (FN) have been reported: one, generated by O‐glycosylation in the splicing region IIICS that is recognized by monoclonal antibody (MAb) FDC‐6, and another, recognized by MAb BC‐I, generated by the alternative splicing of the FN pre‐mRNA which includes an extra type‐III repeat called ED‐B. Using these and 2 other MAbs (IST‐4 which recognizes all different FN isoforms and IST‐6 which recognizes only the FN molecules that do not include the ED‐B sequence) we have immunohistochemically studied 171 normal, hyperplastic and neoplastic breast‐tissue specimens. Although all normal specimens reacted strongly with MAbs IST‐4 and IST‐6, they did not show the presence of oncofetal FNs as established by the use of BC‐1 and FDC‐6. In contrast, out of the 97 cases of invasive ductal carcinomas studied, 90 (93%) and 96 (99%) reacted positively with BC‐I and FDC‐6, respectively, the reaction being observed in the tumoral stroma connective tissue and in tumoral vessels. Furthermore, invasive lobular carcinoma showed less intense and less frequent staining with BC‐1 and FDC‐6 (10 and 11 out of 14, respectively). We found differences in the distribution of the 2 oncofetal fibronectin isoforms within the same specimens. The most remarkable difference was observed in the tumoral vessels: in invasive ductal carcinoma MAb BC‐1 revealed a positive reaction with vessels in 78% of cases while FDC‐6 showed such a reaction in only 59% of cases.
ras‐family gene mutations in neoplasia of the ampulla of vater
Authors: Scarpa Aldo , Zamboni Giuseppe , Achille Alberto , Capelli Paola , Bogina Giuseppe , Iacono Calogero , Serio Giovanni , Accolla Roberto S.
Published: February 2007

Mutations in the first and second exons of Ha‐, Ki‐ and N‐ras oncogenes were investigated in 17 epithelial tumors of the ampulla of Vater by single‐strand conformation polymorphism analysis and direct sequencing of DNA fragments amplified by polymerase chain reaction. The panel included 12 intestinaltype adenocarcinomas, 3 villous adenomas, I papillary carcinoma and I neuroendocrine carcinoma. Six cases (35%) contained ras mutations, affecting codon 12 of Ki‐ras in 2 adenomas and 3 carcinomas, and of N‐ras in I adenoma. All mutations were found in adenomas and among cancers with adenomatous areas, whereas none of the cases lacking adenomatous areas contained mutations. This suggested that ampullary cancers represent heterogeneous diseases with respect to the presence or absence of adenomatous areas and, among those with adenomatous areas, with respect to the presence of activated ras genes. Ki‐ras mutated cases included 3 of 4 tumors which mainly involved the intraduodenal bile duct, thus suggesting that a proportion of Ki‐ras ‐mutated ampullary cancers might correspond to those originating from the epithelium of the bile duct component of the ampulla.
Loss of apc protein expressed by human colonic epithelial cells and the appearance of a specific low‐molecular‐weight form is associated with apoptosis IN vitro
Authors: Browne Sara J. , Williams Ann C. , Hague Angela , Butt Alison J. , Paraskeva Christos
Published: February 2007

APC (adenomatous polyposis coli) protein is differentially expressed in the normal colonic crypt and believed to be involved in colonic cell maturation. In this work we investigated whether expression of the APC protein is associated with cell death in colonic epithelial cells. We have previously reported an in vitro system to study apoptosis. Briefly, cells attached to the flask have a low frequency of apoptosis (1–3%), whereas cells that detach from the flask and float in the medium have a high proportion of apoptotic cells (36–96% depending on the cell line). The full‐length 300‐kDa or truncated APC protein, normally expressed by the attached cells (detected using the FE9 antibody), was found to be lost in the floating apoptotic cells in 8/11 colon tumour cell lines examined. In addition, the APC antibody FE9 detected a 90‐kDa protein in the floating apoptotic cells of all cell lines investigated, which was not present in attached cells. Furthermore, loss of full‐length APC and gain of the 90‐kDa protein was observed in the apoptotic cells of 2 cell lines derived from other tissues: the SV40‐transformed fibro‐blast cell line CMSV40fib and the lymphoblastoid B‐cell line BJA‐B. In cells repeatedly frozen and thawed, believed to induce necrotic cell death, full‐length or truncated APC was also lost, though a 95‐kDa protein distinct from that in apoptotic cells was observed. Specific loss of full‐length or truncated APC (resulting in a 90‐kDa protein in apoptotic cells but a 95‐kDa protein in necrotic cells) is therefore associated with cell death. Our findings suggest a possible role for APC in cell survival.
Inhibition of proliferation and induction of monocytic differentiation in HL60 human promyelocytic leukemia cells treated with bile acids In vitro
Authors: Zimber Amazia , Chedeville Annick , Gespach Christian , Abita Jean‐Pierre
Published: February 2007

We have tested the effect of several bile acids on the proliferation and differentiation of the HL60 human promyelocytic leukemia cell line in vitro. Deoxycholate, chenodeoxycholate and lithocholic acid caused dose‐dependent inhibition of cell proliferation and induction of differentiation along the monocyte/macrophage pathway as determined by morphology, NBT test, non‐specific esterase, and staining by monoclonal antibodies against specific cell‐surface antigens. Optimal effects were obtained at 100, 75, and 60 μM of the 3 bile acids respectively. Cell‐cycle flow‐cytometric analysis showed that a substantial fraction of HL60 cells accumulated at the G0/G1 transition. Protein‐kinase‐C inhibitors such as sphinganine and H‐7 inhibited the differentiation‐inducing effect of bile acids, suggesting a possible role for PKC in this regulation. When bile acids were combined with non‐effective concentrations of all‐trans retinoic acid, enhancement of the monocytic differentiation of THP‐1 human leukemia cells was observed. Our findings demonstrate induction of tumor‐cell differentiation by bile acids, compounds that present minimal undesirable effects in humans.
Ecto‐5'‐nucleotidase (CD73) in multidrug‐resistant cell lines generated by doxorubicin
Authors: Ujházy Peter , Klobušická Margita , Babušíková Olga , Strausbauch Paul , Mihich Enrico , Ehrke1 M. Jane
Published: February 2007

Cytochemical screening for a panel of enzymes revealed increased 5'nucleotidase (5'NT) expression in 3 of 3 Pglycoprotein 170 (Pgp 170)‐positive multidrug‐resistant (MDR) variants of the murine EL4 T‐lymphoma cell line (EL4/ADM, ER2 and ER13). Electron microscopic localization established the presence of the membrane‐bound ecto‐form of the enzyme. Nine other murine, human and Chinese hamster cell lines and their MDR variants were tested for ecto‐5'NT. Of these, 4 MDR variants (human cell lines MCF7A6, MCF7A2, HeLaJ2C and the murine cell line LI2I0A) showed increased expression of ecto‐5'NT, when compared with their parental cell lines. The findings with cells of human origin were confirmed by immunofluorescent localization with a specific monoclonal antibody (MAb) (27.2) against the human ecto‐5'NT. All MDR cell lines with elevated ecto‐5' NT expression were generated by doxorubicin treatment. These cells were more sensitive than their parental cell lines to AMP at concentrations of 1.5–3.0 mM, confirming that the expressed ecto‐5'NT was biologically active. The parental and MDR cells did not differ, in general, in their sensitivity to adenosine. An inhibitor of ecto‐5'NT, α,β‐methyleneadenosine 5'‐diphosphate, completely reversed the resistance of the EL4/ADM cell line to doxorubicin. The possibility exists of a functional relationship between the ecto‐5'NT molecule and the members of the ATP‐binding cassette transporter superfamily, important components of MDR, in some cell types.
Differential intracellular processing of the anthracycline drug ME2303 in doxorubicin‐sensitive (A2780) and ‐resistant (A2780AD) human ovarian cancer cells as studied with confocal laser scanning microscopy and image analysis
Authors: Huxham Ian M. , Barlow Andrew L. , Lewis Alex D. , Plumb Jane , Mairs Robert J. , Gaze Mark N. , Workman Paul
Published: February 2007

Laser scanning confocal microscopy has been used to follow the uptake and efflux of the 2‐fluoroglycoside of doxorubicin, ME2303, in live cultures of the human ovarian cancer cell line A2780 and its doxorubicin‐resistant variant A2780AD. Our methods combine confocal laser scanning microscopy and image analysis to examine the dynamics of anthracycline drugs in cancer cells. Cytotoxicity determined by MTT dye reduction showed that A2780AD cells were more than 400 times less sensitive to doxorubicin compared to A2780 cells but almost 9 times more sensitive to ME2303 compared to doxorubicin. The naturally fluorescent drug was tracked within live cells at 37°C to provide time‐course information in relation to nuclear and Golgi‐associated cellular domains, as indicated by BODIPY FL ceramide‐associated fluorescence. In both cell types, ME2303 was characterised by strong nuclear membrane and peri‐nucleolar fluorescence and as a localised punctate pattern within the nucleus. A2780AD cells accumulated ME2303 in their nuclei at a much reduced rate compared to the doxorubicin‐sensitive cells, and ME2303 efflux from resistant cell nuclei was approximately twice as fast as from A2780 cells. The relative uptake of ME2303 into Golgi‐associated domains and the nucleus were monitored simultaneously during the initial 35 min of exposure to 10 μM ME2303 and during the first 45 min of a “chase” culture following exposure to 20 μM ME2303. ME2303 was detectable within the Golgi‐associated domains of A2780 cells several minutes later and in less relative concentration than in A2780AD cells 15 min into a chase culture. Our results suggest the direct involvement of differences in drug processing via the Golgi apparatus in the expression of P‐glycoprotein‐related drug resistance.
Frequent deletion in chromosome 4 and duplication of chromosome 15 in liver epithelial cells derived from long‐term culture of C3H mouse hepatocytes
Authors: Nishimori Hiroyuki , Ogawa Katsuhiro , Tateno Hiroyuki
Published: February 2007

Prolonged culture of hepatocytes isolated from mouse liver results in the spontaneous development of colonies of liver epithelial cells that can proliferate indefinitely in vitro. We established 5 such cell lines from C3H/HeJ mice (C3H) and 22 cell lines from C3H/HeJ × C57BL/6J F1 mice (C3B6F1) to investigate whether any specific karyotype alterations may be associated with the development of such cells. These lines retained some properties of hepatocytes as well as showing bile‐duct‐cell characteristics, and comprised mainly near‐diploid and/or hypotetraploid cells. Karyotypic analysis of the C3H cell lines indicated that most cells have loss of chromosome 4 or deletion involving the C7 portion, while at least I (for near‐diploid cells) or 2 (for hypotetraploid cells) copies of chromosome 4 were usually intact. In addition, gain of an extra chromosome 15 was frequently observed in these cell lines. Analysis of the microsatellite DNA polymorphic markers in 22 C3B6F1 lines revealed that a majority of them showed loss of heterozygosity (LOH) for, at least, 1 of 3 polymorphic loci on chromosome 4, but not for 2 loci on chromosomes 7 and 11. Mouse chromosomes 4 and 15, therefore, may contain genes related to the ability of such liver epithelial cells to grow indefinitely in vitro [The locus on chromosome 4 was designated as liver‐cell immortalization (LCI) locus].
Alterations in cellular differentiation, mitogenesis, cytoskeleton and growth characteristics during syrian hamster embryo cell multistep In vitro, transformation
Authors: Isfort Robert J. , Cody David B. , Doersen Claus‐Jens , Kerckaert Gary A. , Leboeuf Robert A.
Published: February 2007

In vitro, Syrian hamster embryo (SHE) cell transformation is a neoplastic process that proceeds through several identifiable consecutive stages including in vitro, morphological transformation (mt), acquisition of immortality (I+), acquisition of tumorigenicity (T+) and tumor‐derived cells (I TD). Eight transformed lineages consisting of cells at the mt, I+, T+ and I'TD stages were assayed for alterations in general markers of cell differentiation, mitogenic signaling pathways, cytoskeleton and cellular growth in 3D matrix. Alterations in cellular differentiation markers included a decrease in HI9, gene expression and placental alkaline phosphatase enzymatic activity at the mt stage in all lineages examined with a complete absence of HI9, gene expression and placental alkaline phosphatase enzymatic activity by the I'TD stage in a majority of transformed lineages. Changes in mitogenic signaling pathways included the production of autocrine growth factors and alterations in growth factor‐induced immediate early gene expression by the I'TD stage of transformation in the majority of transformed lineages investigated. By the I'TD stage of transformation in most lineages, changes in both the cytoskeleton (including a decrease in tropomyosin‐1 gene expression) and the Matrigel growth characteristics of SHE cells were observed.
Myeloid and lymphoid cell alterations in normal mice exposed to chemotherapy with doxorubicin and/or the multidrug‐resistance reversing agent SDZ PSC 833
Authors: Froidevaux Sylvie , Loor Francis
Published: February 2007

The cyclosporin SDZ PSC 833 (PSC) is a potent in vivo chemosensitizer for tumor cells with P‐glycoprotein(Pgp)‐dependent multidrug resistance (MDR). However, Pgp expression also occurs in CD8+ T cells, NK cells, macrophages and stem cells. In order to find whether PSC might display specific myelotoxicity or potentiate the toxicity of anti‐cancer drugs, healthy mice were exposed to single doxorubicin (DOX) and combined (DOX + PSC) chemotherapy protocols known to be near or above the borderline of toxicity for tumor‐bearing mice. Mice treated with DOX alone or with (DOX + PSC) showed transient spleen hypoplasia, with a general decrease of all leucocyte lineages and a persistent fall in the numbers of B cells in the bone marrow. In (DOX + PSC)‐treated mice, PSC only potentiated the DOX effects without inducing specific depletions of the Pgp‐expressing leukocytes (CD8+ and Mac‐l+ cells). Hematopoietic cell grafts from normal mice to (DOX ± PSC)‐treated mice did not correct their B‐cell lineage deficiency. When lethally irradiated mice were rehabilitated with hematopoietic cells from (DOX ± PSC)‐treated mice (including those with very reduced survival), all chimeras survived for at least 8 months after the cell graft, at which time their leucocyte population profiles were similar to those of control chimeras.
Expression of the extracellular matrix molecule thrombospondin inversely correlates with malignant progression in melanoma, lung and breast carcinoma cell lines
Authors: Zabrenetzky Vivian , Harris Curtis C. , Steeg Patricia S. , Roberts David D.
Published: February 2007

Thrombospondin (TSP) is a member of a family of extracellular matrix glycoproteins that may participate in multiple aspects of the metastatic cascade. We report an inverse correlation of steady‐state Thbs‐1 mRNA and protein expression with malignant progression among murine melanoma and human lung and breast carcinoma cell lines. Murine K‐1735 melanoma cell lines of low metastatic potential, including K‐1735 lines transfected with the murine nm23‐1 cDNA, expressed higher TSP levels than related highly metastatic lines. In a model system of lung carcinoma malignant progression, immortalized human bronchial epithelial cells expressed higher TSP levels than v‐Ki‐ras, v‐Ha‐ras or n‐ras transfectants, which in turn expressed higher TSP levels than tumor‐derived, more aggressive variants. Among 3 unrelated breast carcinoma cell lines, Thbs‐1 steady‐state mRNA levels were greater in the 2 nonmetastatic lines than the metastatic line. Our data show that malignant progression in some cell lines is associated with reduced TSP expression. The suppressive effects of nm23‐1 transfection on metastatic potential are also associated with increased TSP expression; ras transfection, which results in increased tumorigenesis, is associated with decreased TSP expression.