Fibroblast growth factor receptor 1 is fused to FIM in stem-cell myeloproliferative disorder with t(8;13).

Chromosome 8p11-12 is the site of a recurrent breakpoint in a myeloproliferative disorder that involves lymphoid (T- or B-cell), myeloid hyperplasia and eosinophilia, and evolves toward acute leukemia. This multilineage involvement suggests the malignant transformation of a primitive hematopoietic stem cell. In this disorder, the 8p11-12 region is associated with three different partners 6q27, 9q33, and 13q12. We describe here the molecular characterization of the t(8;13) translocation that involves the FGFR1 gene from 8p12, encoding a tyrosine kinase receptor for members of the fibroblast growth factor family, and a gene from 13q12, tentatively named FIM (Fused In Myeloproliferative disorders). FIM is related to DXS6673E, a candidate gene for X-linked mental retardation in Xq13.1; this defines a gene family involved in different human pathologies. The two reciprocal fusion transcripts, FIM/FGFR1 and FGFR1/FIM are expressed in the malignant cells. The FIM/FGFR1 fusion protein contains the FIM putative zinc finger motifs and the catalytic domain of FGFR1. We show that it has a constitutive tyrosine kinase activity.

t(6;8), t(8;9) and t(8;13) translocations associated with stem cell myeloproliferative disorders have close or identical breakpoints in chromosome region 8p11-12.

A stem-cell myeloproliferative disorder involving T- and B-cell, and myeloid lineages, is associated with three different translocations with a breakpoint in region p11-12 of chromosome 8: t(6;8)(q27;p11), t(8;9)(p11;q33), and t(8;13)(p12;q12), respectively. Using fluorescence in situ hybridization (FISH), we have analysed blood cells from a series of five patients carrying these different translocations. We have identified cosmids from chromosome region 8p11-12 that span the breakpoint in all the cases. They are specific for the FCFR1 gene that encodes a receptor for members of the FGF family. The breakpoint was further detected by Southern and pulsed-field gel electrophoresis analyses with probes from the FGFR1 locus.

Suppression of tubulin tyrosine ligase during tumor growth.

The C terminus of the tubulin alpha-subunit of most eukaryotic cells undergoes a cycle of tyrosination and detyrosination using two specific enzymes, a tubulin tyrosine ligase (TTL) and a tubulin carboxypeptidase. Although this enzyme cycle is conserved in evolution and exhibits rapid turnover, the meaning of this modification has remained elusive. We have isolated several NIH-3T3 derived clonal cell lines that lack TTL (TTL-). TTL- cells contain a unique tubulin isotype (delta2-tubulin) that can be detected with specific antibodies. When injected into nude mice, both TTL- cells and TTL- cells stably transfected with TTL cDNA form sarcomas. But in tumors formed from TTL rescued cells, TTL is systematically lost during tumor growth. A strong selection process has thus acted during tumor growth to suppress TTL activity. In accord with this result, we find suppression of TTL activity in the majority of human tumors assayed with delta2-tubulin antibody. We conclude there is a widespread loss of TTL activity during tumor growth in situ, suggesting that TTL activity may play a role in tumor cell regulation.

Growth modulation of freshly isolated non-Hodgkin's B-lymphoma cells induced by various cytokines and all-trans-retinoic-acid.

We investigated the potential of ten cytokines (IL2, IL3, IL4, IL6, IL10, IL13, G-CSF, GM-CSF, interferon alpha, interferon gamma) and all-trans-retinoic acid to modulate the spontaneous proliferative response in vitro of purified B-non Hodgkin's lymphoma cells of various histological subtypes. 19 malignant lymph nodes were studied. In each case the growth could be influenced by several of these modulators. Cytokines most often implicated were interferon gamma (14/19 cases, 73.7%), IL4 (13/19 cases, 68.4%), interferon alpha (12/19 cases, 63.1%). IL2 (9/19 cases, 47.3%), IL6, IL10, IL13 and ATRA were less frequently involved (6/19 cases, 31.6%) and hematopoietic growth factors (IL3, GM-CSF, G-CSF) were rarely implicated (2/19 cases, 10.5%). The values of growth stimulation ranged from a 1.1-fold to a 6.1-fold increase, and the values of growth inhibition ranged from 15% to 98%. Each cytokine could be either inhibitory or stimulatory depending on the sample analyzed, and no relationship could be found with the histological subtype. Two notable exceptions were IL2, displaying exclusively a positive effect, and ATRA displaying exclusively a negative effect. Overall, these results may have strong implications for future clinical studies using cytokines in the treatment of lymphomas. Ideally, the pattern of in vitro growth response to cytokines or ATRA should be determined individually before undertaking any cytokine treatment.

Effect of oestrone on the natural killer (NK) cell activity, antioxidant status and tumour growth in athymic mice xenografted with human tumours.

Natural killer (NK) cells have been described as being very sensitive to oxidative stress. Thus it has been previously shown that chronic administration of oestrone in drinking water of athymic mice xenografted with a wide variety of human tumours, increases their growth and development. In this study an investigation was made to see whether oestrone supplementation could influence the NK cell activity by changes in the antioxidant defences which result in an oxidative stress and influence the proliferation of tumours. Supplementary oestrone was administered in drinking water of athymic mice xenografted with two different human tumours which lack oestrogen receptors: a bladder carcinoma and a small-cell lung carcinoma. The growth of the urothelial carcinoma was poorly affected by oestrone, but oestrone significantly (p<0.01) increased the proliferation of the small-cell lung carcinoma. The average uterus weight was increased by 62% in oestrone treated mice with no modifications in plasma zinc and selenium status, nor in erythrocyte copper zinc superoxide dismutase level. Nevertheless a slight decrease in erythrocyte glutathione peroxidase activity was noted. Trace elements and antioxidant enzymes in liver homogenates remained unchanged. Oestrone treatment also had no effect on plasma and liver lipid peroxides. The immune response was evaluated by measuring NK activity of splenocytes against 51Cr labelled YAC-I target cells. A 35.5% decrease in the NK activity (p<0.001) was observed after oestrone treatment and may be responsible for graft tolerance. However, the results of these experiments seem to exclude the role of oxidative stress in the modulation of NK activity.

HLA class I and neuroendocrine antigen expression in multidrug resistant small cell lung carcinoma cell lines.

Antigenic marker expression was studied in a series of eight small cell lung carcinoma (SCLC) cell lines, according to their histological subtype, classic or variant. These lines were obtained from human tumors xenografted into nude mice, originally derived from heterotransplanted tumor biopsy samples. We looked at an altered expression of HLA class I antigens, a battery of neuroendocrine antigens and the P-glycoprotein (Pgp) responsible for MDR1 encoded multidrug resistance, as markers of tumor malignancy. Three cell lines out of four of the classic subtype and two cell lines out of four of the variant subtype showed a lack or a low expression of HLA class I antigen. Recombinant interferon gamma (rIFN-gamma) treatment (100 U/ml, for 48 h) increased HLA class I expression of the cell lines differently, but did not induce an imbalance between HLA-A and HLA-B molecules as described in other tumor models. Neuroendocrine antigens were tested in six out of these eight lines, using a family of monoclonal antibodies developed against the cell membrane antigens of low passage cell lines derived from pleural effusions (de Leij et al, Cancer Res 45: 2192-2200, 1985). Globally, these antigens were more highly expressed in classic subtypes of SCLC. Neuroendocrine antigens corresponding to MOC-21 and MOC-32 monoclonal antibodies were weakly expressed in variant forms. Pgp expression was detectable with the JSB1 monoclonal antibody on the three variant SCLCs out of the six lines. Comparing two cell lines originated from the same patient before and after therapy, we showed that neuroendocrine reactivity to MOC-21 and MOC-32 was lost simultaneously with a gain of Pgp expression, and with a classic to variant histological transition. With regard to the clinical evolution, HLA class I expression and stimulation by rIFN-gamma was not related to malignancy. It appears that for variant forms, a low expression of neuroendocrine antigens detected by MOC-21 and MOC-32 monoclonal antibodies and a high level of Pgp predict for a poor prognosis.

Adding a reverser (verapamil) to combined chemotherapy overrides resistance in small cell lung cancer xenografts.

Small cell lung carcinomas (SCLC) are characterised by chemosensitivity to diverse antitumoral compounds. However, responses are transitory and relapses are commonly observed. We examined the ability of verapamil, a reverser of P-glycoprotein (Pgp)-related resistance, to improve the efficacy of CyCAV combined chemotherapy (Cy, cyclophosphamide (CPA); C, cisplatin (CDDP); A, doxorubicin (ADM);V, etoposide (VP16)), as currently administered to SCLC patients at Institut Gustave-Roussy, France, and adapted to the treatment of nude mice implanted with these tumours. Although Pgp encoded by the MDR1 (multidrug resistance) gene is not the only mechanism for multidrug resistance (MDR), and not all drugs included in this regimen are recognised by Pgp, we anticipated a therapeutic benefit. Four different SCLC lines, expressing the MDR1 gene and recently grafted into nude mice, were used. SCLC-75, SCLC-6 and SCLC-41 originated from untreated patients, and SCLC-74T was derived from a patient treated with a combination of ADM, CPA and VP16. SCLC-41% and SCLC-6T tumours were used after having undergone, respectively, five and nine cycles of in vivo passage and CyCAV treatment of the tumour-bearing nude mice, to reinforce their chemoresistance. The efficacy of the CyCAV regimen, associated with or without verapamil (given 24 h before CyCAV on days 1-5), was tested on the growth of these SCLC. Verapamil (25 mg/kg) improved the antitumour effect of CyCAV in mice bearing SCLC-6T, SCLC-41T and SCLC-75 tumours, although toxicity was observed. Verapamil modestly delayed the plasma clearance of ADM. Two daily injections of 10 mg/kg of verapamil, administered at a 3 h interval, proved to be effective, whereas the same total dose administered as a bolus was not. These results indicate that the association of some reversers of MDR, including drugs possibly interacting with Pgp, might potentiate SCLC combined chemotherapy.

Evolution of chromosomal alterations and biologic features in two small cell lung carcinoma cell lines established from one patient during the course of the disease.

Two small cell lung cancer (SCLC) cell lines were established from metastases of a patient during the course of the disease. SCLC 74A was derived from biopsy material obtained at the time of diagnosis and SCLC 74B was from a biopsy specimen of a relapsed tumor obtained after treatment. A transition occurred from SCLC 74A, an intermediate form with 5% large cells to SCLC 74B, a standard mixed form with 20% of large cells, with a decrease in neuroendocrine markers and a substantial increase in P-glycoprotein, a multidrug resistance marker. For both cell lines, R-banding and FISH indicated a del(1)(p35pter) also found in other neural-crest-derived tumors, the loss of regions with suspected tumor suppressor genes at 3p, 5q, and 17p, and a recurrent translocation of the 6q24-6qter region to 10p14. Further genetic modifications in SCLC 74B affected chromosomes 2, 3, 5, 10, 11, 14, and 15. The main observations were a der(2)t(2;5)(p16;q?); a der(3;11)(q10;p10) in SCLC 74A which became der(3;14)(q10;p10) and der(11;14)(p10;q10) in SCLC 74B; and the insertion of the 5q13-5q31 region in the der(10)t(6;10). The finding of the same structural abnormalities in both cell lines suggests a monoclonal origin for both metastases. Hypotetraploid cells were in the same proportion as large cells whose number was a characteristic feature of each cell line. They possessed twice the same chromosomal alterations observed in the hypodiploid cells. This suggests a permanent process of tetraploidization.

Antioxidant status and lipid peroxidation in athymic mice xenografted with two types of human tumors.

Antioxidants and reactive oxygen species are considered to play an important role in experimental in vivo carcinogenesis studies. We attempted in this study to evaluate the repercussions on the antioxidant and lipid peroxide status of the growth of human malignant tumors xenografted into athymic mice. We selected three tumor models: two urothelial carcinomas (bladder tumors stage 3) and one brain tumor (glioblastoma stage 4). All these tumors exhibited a fast growth pattern when xenografted into athymic mice. Tumoral tissue was implanted subcutaneously. After growth establishment each tumor size was measured at regular intervals: every 2 d for bladder tumor and twice a week for glioblastoma. The period of observation was 3 wk for bladder tumors and 5 wk for glioblastoma. At the end of the observation period, all mice were sacrificed; tumoral tissue was taken and blood collected. Superoxide dismutase activity (SOD), glutathione peroxidase activity (GSH-Px), zinc (Zn), selenium (Se), and thiobarbituric acid reactive substances (TBARS) were measured in blood. TBARS alone were measured into tumoral tissue. A modification of the antioxidant blood status was observed in mice xenografted with bladder tumors with decrease in Se status and GSH-Px activities, and increase in TBARS. Such an effect was absent in mice xenografted with glioblastoma. It would appear that an oxygen-mediated stress exists in the animal bearing an implanted tumor compared with the control group, and that tumoral tissue itself is able to induce an oxidative stress into its host. All this leads to a disturbance of the antioxidant defense system.

Response of small-cell lung cancer xenografts to chemotherapy: multidrug resistance and direct clinical correlates.

BACKGROUND: Patients with small-cell lung carcinomas (SCLCs) initially respond to combination chemotherapy. Only a few benefit in terms of long-term survival because most relapse. Such outcome may be attributable to development of multidrug resistance. PURPOSE: The response of SCLC to chemotherapy was examined in terms of (a) patient survival, (b) drug sensitivity of tumors in patients and of tumor xenografts in nude mice, and (c) expression of multidrug resistance gene MDR1 and GST-pi gene. METHODS: Tumor samples obtained from seven untreated patients and from one patient both before and after chemotherapy were transplanted into nude mice. The patients were treated with a combination of cyclophosphamide (C'), cisplatin (C), doxorubicin (A), and etoposide (V) (C'CAV) or C'AV and radiotherapy. Drug sensitivity of SCLCs was tested in nude mice that had received tumor xenografts from these seven patients. The expression of MDR1 and GST-pi genes was assessed in the mRNA extracted from xenografts by Northern blot analysis. P-glycoprotein was quantified by enzyme immunoassay. RESULTS: The patients' responses to C'CAV closely correlated with those of the corresponding xenografts. The tumors of the two patients who showed long-term survival after C'CAV completely regressed when they were transplanted into nude mice and subsequently treated with C'CAV. Despite initial complete response, the remaining five patients died during year 1. A high percentage of mice receiving the tumor grafts from these five patients showed only partial tumor regression after C'CAV treatment. The MDR1 transcript was detected in all five of these xenografts. Four of five xenografts were from untreated patients, and the fifth was from a treated patient. MDR1 mRNA expression was absent in the tumor of this fifth patient before chemotherapy, but both the mice receiving the corresponding xenograft and the patient showed expression of MDR1 after C'CAV treatment. MDR1 mRNA expression was absent in the tumor xenografts obtained from two patients with long-term survival. Expression of P-glycoprotein correlated with MDR1 mRNA expression. All xenografts except one expressed the GST-pi gene. CONCLUSIONS: The absence of MDR1 gene expression during chemotherapy for SCLC indicates a favorable prognosis, gene expression is often coincident with ineffective chemotherapy, and tumor xenografts can be appropriately used to predict response to chemotherapy. IMPLICATIONS: Failure of chemotherapy to control SCLC seems to be related to an acquired multidrug resistance involving the MDR1-mediated mechanism. Therapeutic benefit could therefore be expected from chemotherapy combined with inhibitors of MDR1.

Immunohistochemical study of p53 in human lung carcinomas.

Immunohistochemical analysis of p53 protein was carried out on 95 lung carcinomas from all histological types, including 60 primary tumors, 35 lymph node metastases, and 36 corresponding nude mice xenografts, using four antibodies: PAb240 specific for some mutant conformations; PAb421, PAb1801, and CM1 reactive with most of the forms of p53. Nuclear staining with at least two of those four antibodies revealed the presence of an accumulated protein, considered as indicative of a missense mutation in the p53 gene, in 50% of primary tumors of all histological types, except carcinoids. Some defect of messenger RNA expression was detected by Northern blot analysis in an additional 26% of tumors. p53 immunophenotype of the original tumor was fairly maintained on nude mice. p53 accumulation was not correlated with survival, but with disease extension (P = 0.01). Finally, immunohistochemical analysis allowed the recognition of p53 mutant immunophenotype in 41% of tumors where p53 DNA and messenger RNA were apparently normal, using standard molecular biology. Thus, this method provides a rapid and efficient approach for studying p53 mutations leading to an accumulated protein in lung tumors cells.

Antitumoral activity of bombesin analogues on small cell lung cancer xenografts: relationship with bombesin receptor expression.

Gastrin releasing peptide (GRP), the human homologue of bombesin (BN), is an autocrine growth factor for small cell lung cancer (SCLC) cells. The synthetic octapeptides [D-cpa1-beta-Leu8-des-Met9]litorin (BIM 26182) and [D-Phe6-Leu13-CH2NH-Cpa14]bombesin(6-14)NH2 (BIM 26189) are potent GRP/BN antagonists of the proliferation of 3T3 and rat pancreas cells. The effect of these analogues on the proliferation of four SCLC cell lines (SCLC 6, SCLC 41, SCLC 75, SCLC 74R) was tested in vitro and in vivo. Two of these SCLC lines (SCLC 41M and SCLC 75) had receptors for BN/GRP and expressed the prepro-GRP mRNA. BIM 26182 and BIM 26189 inhibited [3H]thymidine incorporation into the DNA of SCLC 41 cells, stimulated [3H]thymidine incorporation in SCLC 6, and had no effect on the two other cell lines. The SCLC implanted s.c. in nude mice were treated with either BIM 26182 or BIM 26189. BIM 26182 and BIM 26189 injected at the doses of 50 micrograms twice a day (s.c.) around the tumor for 10 to 21 days delayed the growth of SCLC 41 and of SCLC 75. The maximal effect was observed during the treatment period, after which the tumors regrew, suggesting a cytostatic effect of these peptides. No inhibitory effect of the peptides on SCLC 74R or SCLC 6 growth was observed. These data suggest that GRP antagonists are able to inhibit the in vitro and in vivo growth of BN/GRP receptors-positive SCLC.

Effects of free and liposome-encapsulated taxol on two brain tumors xenografted into nude mice.

Free taxol and liposome-encapsulated taxol were compared for their antitumoral activities on two human brain tumors serially grafted into female athymic mice in the scapular region. In the first experiment, a human glioblastoma (15th and 16th passages) was studied. In the second experiment, a fast growing human gliosarcoma (19th passage) was used. Free taxol and liposomal taxol were administered intraperitoneally, at the same dose; 12.5 mg/kg (i.e. 1/15 of the evaluated LD 50 value). In the first experiment, the treatment was performed for four consecutive days, with four courses separated by three rest periods of three days in between. Both free taxol and encapsulated taxol produced a statistically significant delay in tumor growth, and at the end of the experiment some total tumor regressions were obtained. However, liposomes were observed to be more effective in their action on the two consecutive passages of the glioblastoma, giving a marked increase of the number of total tumor regressions. In the second experiment another schedule of treatment was chosen because of the fast growth pattern of the xenografted human gliosarcoma: free taxol and liposome-encapsulated taxol were administered for five consecutive days and three courses of treatment were performed with two rest periods of two days. The two forms of taxol had a significant inhibitory effect on gliosarcoma tumor growth; as before encapsulation in liposomes was found to increase the anti-tumoral activity of taxol, although, in this case no tumor regression was observed.

Activation of myc gene family in human lung carcinomas and during heterotransplantation into nude mice.

myc gene family activation (c-myc, L-myc, and N-myc) was examined in 26 human lung carcinomas and in their corresponding xenografts in nude mice. Of the 16 neuroendocrine (NE) carcinomas studied, amplification was observed in 4 with a c-myc probe and in 1 with both L- and N-myc probes. Overexpression was found in 1 of 7 cases studied for c-myc mRNA, in 1 of 7 cases for N-myc, and in 2 of 7 cases for L-myc. Of the 10 non-small cell lung carcinomas studied, only c-myc was amplified in 1 case and overexpressed in 5 of 7 cases. These results suggest that L- and N-myc gene activation are restricted to NE carcinomas. Over-expression of the myc gene without amplification was detected in 36% of cases. During heterotransplantation, there was a 27% change in myc gene abnormality and a 57% increase in myc expression levels, mostly in NE carcinomas (5 of 7; 71%). In a total of 42 xenografted lung carcinomas studied, 45% amplification and 77% overexpression of one of the myc genes were detected with a high prevalence of L-myc overexpression in NE carcinomas (50%) and of c-myc overexpression in non-small cell lung carcinomas (66%). Finally, 19 of 26 (73%) tumors are growing in nude mice with no myc gene amplification and 43% with no myc mRNA overexpression. Thus myc gene activation is not strictly required for heterotransplantation but seems to be a favorable factor in the maintenance and progression of lung carcinomas in vivo.

Cytotoxic chemotherapy induces cell differentiation in small-cell lung carcinoma.

Despite the high response rates resulting from chemotherapy, the majority of small-cell lung cancer (SCLC) patients relapse with chemoresistant tumors. To analyze the phenotypic changes that are precursors of chemoresistant status, and to investigate the role of chemotherapy in these changes, tumor samples from 20 patients, taken before chemotherapy (etoposide, doxorubicin, and cyclophosphamide) and again at the onset of chemoresistance (after at least three courses of chemotherapy), were compared. The histologic changes were minor in 10 of 20 patients, as shown by an increase in cell size; they were major in 10 of 20 patients, with the appearance of mixed composite tumors in which neuroendocrine (NE), epidermoid, and glandular components were mixed. Major changes correlated with a good response to chemotherapy (P = .001). Ultrastructural studies showed an increase in neurosecretory granules and desmosomes, and a high frequency of multidirectional differentiation (45%) when comparison was made with pretherapy samples (10%) (P less than .01). Immunohistochemical (IH) analysis showed an increase in cytokeratin (CK) expression in treated patients, with a different labeling pattern and the expression of higher molecular weight CK. The expression of NE lineage markers (Leu 19, Sy 38, SL 11-14) remained stable, while that of NE differentiation markers (Leu 7, chromogranin) increased in the treated patients. The neuron-specific enolase (NSE) activity remained stable in treated SCLC. Large cells with a more differentiated phenotype and proliferative capacity (as shown by Ki 67 labeling), appeared to be characteristic of treated and secondary chemoresistant SCLC. The acquisition of a more complex phenotype, which correlates with primary response to therapy, implies a drug-induced differentiation in SCLC.

UV-induced squamous cell carcinomas in the hairless mouse. Morphological characteristics and transplantation in the syngenic and nude mouse.

Squamous cell carcinomas were induced by UVB in the hairless mouse HRO hr/hr. Twelve mice were irradiated three times a week at a dose of 0.19 J/cm2. The average latency period for the appearance of the first tumour is 16 weeks and by the 21st week, tumour incidence is 100%. Further observation of these tumours shows that their growth is independent of irradiation dose and that the tumours continue to appear even after cessation of irradiation. Histological and cytological examination of these tumours show them to have characteristics of malignancy. Transplantation in athymic mice suggests that UVB provokes immune deficiencies in hairless mice similar to those seen in conditioned nude mice.