Establishment of two new human bladder carcinoma cell lines, CAL 29 and CAL 185. Comparative study of cell scattering and epithelial to mesenchyme transition induced by growth factors.

We describe here two new human urothelial carcinoma cell lines, CAL 29 and CAL 185, established from two patients with high-grade tumours and which display very different properties in vitro. We have shown that CAL 29 cells were tumorigenic in mice and expressed characteristic features of both cell scattering and transition from epithelial to mesenchymal phenotype (EMT) after triggering by the EGF receptor ligands, TGFalpha and EGF. At the opposite, the CAL 185 cells were not tumorigenic in mice and neither scattered nor expressed vimentin intermediary filaments in the presence of growth factors. We further demonstrated that CAL 29 cell scattering was reversible after growth factor removal and that both scattering and EMT were markedly impaired after treatment with MEK, Src and PI3-kinase inhibitors suggesting that these kinases might be important components of the cellular responses to EGF and TGF-alpha leading to scattering and EMT. These agents could help to understand the intracellular pathways involved in invasiveness and to find new targets for limiting metastasis. In conclusion, these two new cell lines could be good models to dissect the molecular mechanisms involved in invasion and metastasis development in human bladder cancer.

Screening of human bladder carcinomas for the presence of Ha-ras codon 12 mutation.

Contradictory results were obtained from previous studies aiming at defining the frequency of Ha-ras codon 12 mutations in bladder tumors. Differences in the sensitivities of the methods used could account for this discrepancy. In this study, we reevaluated the frequency of Ha-ras codon 12 mutations in a series of 87 human bladder tumors using a combination of two different methods. The first was derived from the protocol of Ooi et al and consisted in a one-step allele-specific polymerase chain reaction using mismatched primers in two separate PCR. This method is very rapid and highly sensitive, detecting the presence of minor populations (less than 10%) of mutant alleles. The second strategy consisted in screening all tumors using natural restriction fragment length polymorphism (RFLP) analysis. The two methods were in complete concordance and enabled us to show that only one out of 87 primary bladder carcinomas (1%) exhibited the mutation, in accordance with previous studies. These results strongly suggest that, even if minor cell populations overexpress codon 12 Ha-ras mutation, the analysis of this mutation cannot be used to screen potentially invasive transitional cell tumors of the bladder.

Expression of vascular endothelial growth factor-b in human astrocytoma.

Growth of human malignant gliomas is stringently dependent on an angiogenic process that probably involves vascular endothelial growth factor (VEGF). Expressions of mRNA coding for the different forms of VEGF were analyzed in surgical specimens from human astrocytomas. Low levels of placental growth factor (PGF) and VEGFC mRNA were observed in polymerase chain reaction, but not in Northern blot experiments. VEGF mRNA was found in some but not all grade and grade IV astrocytomas. VEGFB mRNA was observed in all tissue samples analyzed irrespective of the tumor grade. A new splice variant of VEGFB (VEGFB155) that lacks exons 5 and 6 is described. Expressions of VEGF mRNA in cultured glioblastomas cells were upregulated by hypoxia, but the sensitivity of the cells to hypoxia was reduced as compared with normal rat astrocytes. VEGF expression was depressed by dexamethasone. Expressions of VEGFB mRNA were affected neither by hypoxia nor by dexamethasone. The results indicate a coexpression of VEGF mRNA and VEGFB mRNA in human astrocytomas. Expression of VEGFB is markedly different from that of VEGF. Possible roles of VEGFB as a cofactor for hypoxia-induced angiogenesis in human astrocytomas are discussed.

Establishment, characterisation and partial cytokine expression profile of a new human osteosarcoma cell line (CAL 72).

Permanent human osteosarcoma cell lines are important tools for the study of bone cancer. As representative of an osteoblastic phenotype, they partly reflect their normal osteoblastic counterparts and, thus, may represent appropriate models to investigate the mechanisms involved in bone remodelling and in haematopoietic differentiation. In the present work, we describe a new human cell line, CAL 72, obtained from an osteosarcoma of the knee of a 10-year-old boy. These cells grow in continuous culture, and karyotypic analysis has revealed clonal abnormalities in number and structure, especially loss of chromosome Y. These cells exhibit morphological, immuno-histochemical and molecular characteristics of the osteoblastic lineage. Using RT-PCR, we have shown that the CAL 72 cell line expresses high levels of mRNA coding for several cytokines, such as G-CSF, GM-CSF, IL-1beta and IL-6. In view of this expression profile, the CAL 72 phenotype appears to be closer to normal primary osteoblasts than other reported osteosarcomas. Moreover, these cells express mRNA for both HGF and its receptor c-MET, suggesting that this autocrine loop might contribute to the invasiveness of the tumour from which CAL 72 originated.

Human breast-cancer metastasis formation in a nude-mouse model: studies of hyaluronidase, hyaluronan and hyaluronan-binding sites in metastatic cells.

Few animal models are available to study metastasis formation. The purpose of the present study was to obtain a useful model of metastasis formation in nude mice in an attempt to analyze the stroma reaction and in particular the production and the expression of hyaluronan (HA), hyaluronidase, and HA-binding sites by cultivated cells, and HA and hyaluronectin (HN) in the invasive areas of tumors. Nude mice were subjected to i.p. injections of several human cancer cell lines (PLC/PRF/5, HepG2, CB 191, CB 193, PC3, CAL 51, SA 87 and SA 98), and formation of metastases was analyzed in different organs (lung, liver, kidney, spleen and axillary nodes) by immunohistochemical techniques. CAL 51, a breast-cancer-metastasis-derived cell line with a normal karyotype, produced i.p. tumors in 75% animals and metastases in 90% animals (detected in the liver and axillary nodes). Two modes of invasion by CAL 51 cells were observed in the liver: one, direct, from the surface of the liver and the other, indirect, via the bloodstream. HA and HN were strongly expressed at the invasion areas. A cell line derived from hepatic metastasis of CAL 51 (HMD CAL 51) presented an abnormal karyotype. HMD CAL 51 produced more hyaluronidase (12-fold) and HA (10-fold) and expressed more CD44 (1.6-fold) and other HA-binding sites (9.5-fold) than the established cell line CAL 51. Our results show that i.p. injection of the CAL 51 cell line into nude mice provides a useful model of metastasis formation. The passage of the CAL 51 cells from the primary state to the metastatic state was characterized by a dramatic increase of HA and hyaluronidase production, and expression of HA, HN and HA-binding sites.

Epidermal growth factor receptor and labeling index are independent prognostic factors in glial tumor outcome.

The aim of this study was to perform a multivariate analysis including clinical and biological prognostic factors on glial tumor outcome. Seventy-nine patients were analyzed (48 men and 31 women; mean age = 56 years, range = 16-77 years): 7 had a benign glial tumor (grades 1 and 2), 21 had an anaplastic glial tumor (grade 3), and 51 had a glioblastoma (grade 4). Median follow-up was 17.9 months for patients who survived (50 patients died). Biopsies were obtained at time of diagnosis (complete tumor resection in 62 patients and stereotaxic biopsies in 17 patients). Epidermal growth factor receptor (EGFR) was measured by a binding assay, and labeling index (LI) was measured by tritiated thymidine incorporation. EGFR varied from 4 to 73,110 fmol/mg protein (mean = 3912 fmol/mg protein; median = 374 fmol/mg protein; n = 79). LI varied between 0.1 and 16.5% (mean = 6.2%; median = 5.2%; n = 40). Log10 EGFR was significantly and positively correlated with patient age. LI was significantly different according to tumor histology. Univariate Cox analysis (end point was cancer death) showed that age (P = 0.027), log10 EGFR (P = 0.025), and LI (P = 0.0019) were significant continuous variables, the survival being shortened when the covariable increased; tumor resection (P = 0.015, relative risk = 0.45) and histology (P = 0.0009) were significant categorical factors. A multivariate Cox analysis (forward selection) including age, histology, tumor resection, log10 EGFR, and LI revealed that log10 EGFR, LI, and tumor resection were the only independent significant predictors of survival. This multivariate approach reveals that the clinical prognostic factors of glial tumors, namely age and tumor histology, disappear, to the benefit of intrinsic characteristics of the tumor, i.e., EGFR expression and LI, suggesting that coupled EGFR and LI determination could be a useful tool for better evaluation of glial tumor outcome.

Non-specifically labelled cells that simulate bone marrow metastases in patients with non-metastatic breast cancer.

AIMS: To determine whether the presence of disseminated bone marrow tumour cells at diagnosis is a prognostic factor for breast cancer patients at high risk of recurrence or bone metastasis, and to assess their presence as a criterion for evaluation of the potential benefits of adjuvant chemotherapy. METHODS: Multiple bone marrow aspirates from 72 breast cancer patients free from metastasis were obtained during surgery at the time of diagnosis and were tested immunologically by alkaline phosphatase antialkaline phosphatase technique with a panel of three antiepithelial monoclonal antibodies (MoAb) KL1, EMA, and HMFG2. RESULTS: In nine of 72 patients, with each MoAb tested, numerous strongly positive cells always isolated were observed. However, it was demonstrated that these cells were non-specifically labelled and could be found in normal controls. CONCLUSIONS: There was no evidence of marrow tumour cells in 72 operable breast cancer patients. It is suggested that published results may be greatly overestimated and that non-specific labelling may be undetected. More specific MoAb should be found and a correlation with molecular biology should be performed if this criterion is to be considered as a prognostic factor.

[GC12, marker of cells of mesodermal origin. Value and application to cytodiagnosis of serous effusions]. / GC12, marqueur des cellules d'origine mésodermique. Intérêt et application au cytodiagnostic des épanchements de séreuses.

Monoclonal antibody GC12 was examined for its value in cytopathologic diagnosis. Its sensitivity and specificity in staining reactive mesothelial cells in serous effusions was determined using the indirect immuno alkaline phosphatase anti-alkaline phosphatase technique. Smears prepared from 78 serous effusions (15 benign, 54 malignant, 9 atypical) were immunostained with GC12 and five other monoclonal antibodies: KL1 directed against cytokeratins, D33 against desmin, E29 against epithelial membrane antigen, Calam 27 against epithelial surface antigen and NEO 723 reactive with carcinoembryonic antigen. GC12 reacted positively with 80 to 100% mesothelial cells from the 15 benign effusions. Carcinoma cells were negative in 87% of malignant cases. Ovarian carcinomas were specially stained among positive cases. One malignant mesothelioma was positive and one was unreactive with GC12. Moreover, the reactivity of atypical cells in 9 serous effusions with GC12 was helpful to characterize benign and malignant effusions: the immunophenotype GC12-, Desmine-, Calam 27+, EMA+, ACE+ allowed detection of carcinoma cells in 2 cases. It is concluded that GC12 is a good tool for distinguishing carcinoma cells from reactive mesothelial cells in serous effusions.

Establishment and characterization of a new cell line derived from a human chondrosarcoma.

A new human cell line, CAL 78, derived from a dedifferentiated chondrosarcoma of the muscle of the thigh has been established in culture. Fibroblastoid morphology, vimentin expression and lack of epithelial antigens are in agreement with mesodermic origin of these cells. The xenograft of CAL 78 cells in nude mouse showed the characteristics of hyaline cartilaginous differentiation. Cytogenetic changes were numerous and complex, all the metaphases were tetraploid and no alterations described by other authors have been found. CAL 78 constitutes an appropriate model to evaluate efficiency of a new therapy for chondrosarcomas. Moreover, this cell line may be used to study some stage of chondrocytic differentiation.

[Production of hyaluronidase by cultured human tumor cells]. / Production d'hyaluronidase par les cellules cancéreuses humaines en culture.

The presence of hyaluronidase was detected at pH 3.8 in eight out of twelve human cancer cell line culture media. Eight cell lines derived from primary tumours and four from metastases. In three culture media the enzymatic activity was lower than 0.035 pU/cell/h. In five others (in a hepatoma cell line and in four metastasis-derived cell lines) the activity was higher than 0.057 pU/cell/h. A tumour-derived fibroblast culture was negative. The optimal activity was observed at a pH comprised between 3.6 and 4. Salt inhibition of hyaluronidase was reversible. The enzyme was denaturated by a 10-min heating at 70 degrees C. The enzyme was not strictly specific for hyaluronan hydrolysis but also digested chondroitin sulfates. PH20, a spermatozoid protein that has homologies with the bee venom hyaluronidase, was not expressed by cell lines tested.

[CAL 54, a new cell line derived from a human renal carcinoma: characterization and radiosensitivity]. / CAL 54, une nouvelle lignée cellulaire dérivée d'un carcinome rénal humain: caractérisation et radiosensibilité.

A new cell line (CAL 54) was isolated from a malignant pleural effusion of a patient with renal carcinoma. CAL 54 is a continuous and stable cell line. Immunochemical staining showed simultaneous expression of cytokeratin, epithelial membrane antigen and vimentin. Cytometric flow analysis of DNA content reveals one major hyperdiploid population. Histological aspect of the tumor induced in the nude mouse showed well differentiated adenocarcinoma with papillary structure. Radiation response of these cells was evaluated by the colony-forming method and the data were fitted with the linear-quadratic model. Survival at 2 Gy (SF2) was 0.28 and the mean inactivation dose (D) = 1.50 Gy, ranking this cell line among the radiation sensitive cells. CAL 54 may be an informative cell line to investigate radiation effects in the management of renal tumours.

Micronucleus induction in 10 human tumour cells after high- and low-dose radiation.

A number of data measuring survival of animal or human cells to low LET ionizing radiation have demonstrated that these cells may be hypersensitive to doses below 1 Gy, possibly due to the absence of an inducible repair mechanism, which is observed at higher doses. The production of micronuclei (MN) in cells exposed to ionizing radiation reflects genotoxic damage. Moreover, the micronucleus assay is sensitive to low radiation doses. We have exposed 10 human tumour cell lines to doses ranging between 0.12 and 4 Gy. Using cytochalasin B to block the cells in a binucleate phase, we have scored the fraction of binucleate cells (BNC) expressing MN, as well as the number of MN per BNC, as a function of gamma-ray dose. Experimental points were fitted with a binomial equation. Doses from 1 to 4 Gy were fitted separately from those below 1 Gy, and the initial slopes after both fits were compared. Taken together, the initial slopes of MN induction after low-dose (LD) irradiation were not different from those after high-dose (HD) irradiation. Only in one cell line was a significant increase in MN production detected after LD irradiation. This cell line had the shallowest linear term after HD irradiation. It appeared that the likeliness of expressing hypersensitivity at LD was correlated with the quadratic term of MN induction at HD, which does not contradict an inducible repair hypothesis. However, the failure of observation of a significant hypersensitivity at LD for nine cell lines, and the high variability of response at LD suggests that this occasional effect may be influenced by other factors as well.

Cytogenetics of malignant gliomas: I. The autosomes with reference to rearrangements.

Autosomal chromosome abnormalities are far from always detectable and, when detected, far from fully consistent in malignant gliomas. In 15 of 41 malignant gliomas, we found autosomal chromosome aberrations ranging from solitary trisomy to a wildly abnormal polyploid complement. The sequence of chromosome events appears to proceed from the normal to the near-diploid state (via structural and numerical changes) to near-tetraploidy (via polyploidization), and finally toward near-triploidy (via chromosome loss and additional rearrangements). Characteristic chromosome changes of trisomy 7 and monosomy 10 were repeatedly found, usually together in the same cell clones. In only one case was trisomy 7 an isolated change. We observed structural rearrangements of chromosomes 7 and 10 which may be of some use in mapping specific genes duplicated or deleted by the whole-chromosome changes of chromosomes 7 and 10. Nonrandom structural changes of other autosomes, including chromosomes 1, 5, and 11, fit with the model of malignant glioma as a process involving multiple genes. An unusual concentration of breakpoints in 12q13, juxtaposing it to at least five other regions, reflects the presence of genetic information in 12q13 important to the development of malignant gliomas.

Cytogenetics of malignant gliomas. II. The sex chromosomes with reference to X isodisomy and the role of numerical X/Y changes.

Sex chromosomal monosomy with total loss of an X or Y is frequently observed in malignant gliomas. Beyond that, not much is known about the behavior of the sex chromosomes in these tumors. We noted loss of the X from 3 of 13 gliomas from women (23%) compared to loss of the Y from 16 of 28 gliomas from men (57%). There were two structural rearrangements of the Y (an inversion and a translocation with chromosome 4). Most unexpectedly, clones with sex chromosome reversal were encountered in 3 cases. These XX clones in gliomas from men are perforce the consequence of Y loss coupled with X isodisomy, a nonrandom sequence of sex chromosome changes. We examined the company kept by numerical X and Y changes in clones and found that clones with numerical sex chromosome changes had fewer autosomal abnormalities, reflecting a distinct tendency to clonal separation of sex chromosome from autosomal abnormalities. We conclude that the sex chromosome changes are not a necessary part of the neoplastic process in malignant gliomas but that they must be of biologic significance to the brain since they are highly nonrandom in frequency, type, and sequence in brain cells.

Chromosomes in gliomatosis cerebri.

Gliomatosis cerebri is a rare brain tumor which histologically resembles a diffuse cerebral astrocytoma. It can simultaneously infiltrate multiple sites in the cerebrum, cerebellum, brainstem, and spinal cord. This remarkable diffuseness has led to the idea that gliomatosis cerebri does not derive from a solitary focus but must arise from a broad field of glial cells. We studied the chromosomes from gliomatosis cerebri in a 12-year-old boy by conventional cytogenetics and fluorescence in situ hybridization (FISH). Aside from normal cells, we found a majority of cells with the karyotype 44,XY,del(6)(q25),del(14)(q21), der(15;21)(q10;q10),add(18)(q22),del(19)(p12),add(20)(p13),-21. A smaller proportion of cells had 88 chromosomes with a doubling of this abnormal karyotype. These findings are consistent with a clonal neoplasm stemming from a single cell. The chromosome changes we observed, with the possible exception of the chromosome 6 deletion, did not resemble those frequently found in astrocytomas. Gliomatosis cerebri may therefore belong to a separate category of brain tumors.

Immunohistochemical determination of nuclear antigens by colour image analysis: application for labelling index, estrogen and progesterone receptor status in breast cancer.

The immunohistochemical evaluation of 5-bromo-2'-deoxyuridine (BrdU) labelling index, estrogen (ER) and progesterone receptor (PR) status was carried out on the automated computer-assisted image analysis station BIOCOM 500. Special software has been developed to measure nuclear antigens using the immunoperoxidase method with the Harris hematoxylin counterstain. The analysis was based on the different light adsorption spectra of the chromogen diaminobenzidine and Harris's hematoxylin coloration when exposed to light of differing wavelengths. The results obtained by image analysis were compared to previously validated methods. The thymidine labelling index performed by manual procedures and BrdU incorporation performed by image analysis were comparable (linear correlation coefficient r = 0.76, P < 0.001). Comparison of image analysis and dextran coated charcoal assay for ER and PR content revealed excellent sensitivities and specificities (linear correlation coefficient r = 0.87, P < 0.001 for ER and r = 0.93, P < 0.001 for PR). These data suggest that automated image analysis offers a reliable and reproducible procedure for measuring nuclear antigens.

Immunophenotyping and metastatic localization of breast cancer.

Monoclonal antibody SM 92 is involved in the immunophenotype of gastrointestinal and liver cells, SM 43 in ovarian cells, and SM 13 in lung cells. Based on a study of 61 breast adenocarcinoma patients, we found that tumors reacting with SM 92 appear associated with liver metastases, SM 43 with ovarian metastases, and SM 13 with lung metastases. These associations are highly significant. They lend some support to the concept that tumor cells that metastasize tend to go to sites where cells normally have the same surface antigens.

Effects of tamoxifen on potential doubling time of human breast cancer cell line determined by image cytometry of double fluorescent BrdU and DNA labeling.

Tamoxifen is extensively used for the treatment of human breast cancer. However, the mechanisms by which antiestrogens regulate the growth of estrogen receptor positive tumors have not been totally defined. A new methodology, using automated image analysis BIOCOM 500, was developed for determining potential doubling time (Tpot) of tumors. This new method was checked on three different human breast cancer cell lines (MCF-7, CAL 85-1, CAL 148) in comparison with flow cytometry and then applied to determine the effects of short-term tamoxifen treatment on Tpot of MCF-7 cells. Using the resulting bivariate contour plot of blue fluorescence (DNA content) versus green fluorescence (Bromodeoxyuridine content), a labeling index (LI) value of 0.39 +/- 0.05 and a Tpot value of 21 +/- 2.09 hours were determined for MCF-7 cells. As expected, data demonstrated that 72 hours of 1 microM tamoxifen treatment decreased the LI to 35% by increasing the proportion of G0/G1 cells. It increased the Tpot to 35% compared to untreated cells (Tpot = 31.8 + 4 hours) by a lengthening of G0/G1 phase without changing the length of S phase (Ts = 10.2 +/- 1 hours). At suprapharmacological concentrations (5, 10 microM), an approximately 50% increase in Tpot was observed without modification in Ts. These data suggested a specific cell cycle action of tamoxifen which was probably mediated by mechanisms other than estrogen inhibition, since these experiments were performed in estrogen-deprived medium. In addition, the automated imaging procedure appears to provide a rapid and quantitative approach to determine Tpot in fine needle biopsies which is useful for investigating alterations in cell growth after endocrine treatment or chemotherapy.

[Forum on tissue expansion. Tissue expansion and radiotherapy. Changes in epidermal mitotic index induced by expansion and/or irradiation in the rat]. / Forum: l'expansion tissulaire. Expansion tissulaire et radiothérapie. Variations de l'activité mitotique épidermique induites par expansion et/ou irradiation, chez le rat.

In order to study the influence of irradiation during skin expansion, an experimental protocol using 15 laboratory rats was undertaken. The present experiment was designed to evaluate the mitotic activity of the epidermis by in vitro autoradiography. The mitotic activity was assessed by the Labelling Index. Experimental data concerning expansion were shown to be consistent with the literature. When a high dose of radiation was delivered to the skin during expansion, a significant increase was observed in the mitotic activity of the epidermis. The range of variation was equivalent to that expected in normal skin. This is the first experimental result in favour of irradiation during skin expansion. However, no conclusion can be drawn concerning the clinical applications (immediate breast reconstruction using tissue expansion) due to the limitations of the present experiment.

Characterization of cal39, a new human cell-line derived from a vulvar squamous-cell carcinoma.

We have obtained a permanent cell line from a squamous cell carcinoma of the vulva. These cells, named CAL39, exhibited morphological, ultrastructural, and immunochemical characteristics of epithelial cells. They were tumorigenic in nude mice. Our observations indicate that the behavior of the cell line in the nude mouse and in culture is similar to that of the original cancer from which it was derived. We have compared these cells with A431 which are currently the best studied model cells of the same tumor origin. Like A431, CAL39 had an elevated number of high affinity EGF receptors, and showed an amplification of DNA sequences at 11q13. We have compared the cytogenetic features of the amplification units in the two cell lines. Unexpectedly, an HSR was present, in both cases, on a marker chromosome which was a derivative of chromosome 11. This result addresses the issue of the in situ amplification of chromosomal DNA.