A phase II trial with mitozolomide was carried out in patients with malignant melanoma, since in ... more A phase II trial with mitozolomide was carried out in patients with malignant melanoma, since in preclinical studies this new imidazotetrazine had shown promising effects against human melanoma xenografts. Twenty-one evaluable patients with advanced malignant melanoma were teated with 115mgm-2 of mitozolomide, given orally every 6 weeks. None of the patients had received prior chemotherapy. Two partial responses (10 and 7+ months) were observed. The responding patients had lung metastases, and one of them had, in addition, a huge (17 x 14 cm) lymph node metastasis in the groin. Also, one patient had a 48% tumour volume reduction of lung metastases. The dose limiting side effect of the treatment was bone marrow depression, with delayed leukopenia and thrombocytopenia. The median white blood cell counts and platelet nadirs were 2.5 x 109 1-1 (range 1.1-3.8) and 59 x 109 11 (range 14-95), respectively. Nonhaematological adverse reactions were limited to mild or moderate nausea. It is concluded that orally administered mitozolomide is active against malignant melanoma and seems to have a response rate comparable to those of the most active established drugs.
In a recent comparative genomic hybridization (CGH) study of a panel of sarcomas, we detected rec... more In a recent comparative genomic hybridization (CGH) study of a panel of sarcomas, we detected recurrent amplification of 1q21-q22 in soft tissue and bone tumours. Amplification of this region had not previously been associated with sarcoma development, but occasional amplification of CACY/S100A6 and MUC1 in 1q21 had been reported for melanoma and breast carcinoma respectively. Initial screening by Southern blot analysis showed amplification of S100A6, FLG and SPRR3 in several sarcomas and, in a first attempt to characterize the 1q21-q22 amplicon in more detail, we have now investigated the amplification status of these and 11 other markers in the region in 35 sarcoma samples. FLG was the most frequently amplified gene, and the markers located in the same 4.5-Mb region as FLG showed a higher incidence of amplification than the more distal ones. However, for most of the 14 markers, amplification levels were low, and only APOA2 and the anonymous marker D1S3620 showed high-level amplifications (> tenfold increases) in one sample each. We used fluorescence in situ hybridization (FISH) to determine the amplification patterns of two overlapping yeast artificial chromosomes (YACs) covering the region between Dl S3620 and FLG (789f2 and 764a1), as well as two more distally located YACs in nine selected samples. Six samples had amplification of the YAC containing DlS3620 and, in three, 764a1 was also included. Five of these tumours showed normal copies of the more distal YACs; thus, it seems likely that an important gene may be located within 789f2, or very close. Two samples had high copy numbers of the most distal YACs. Taken together, FISH and molecular analyses indicate complex amplification patterns in 1q21-q22 with at least two amplicons: one located near Dl S3620/789f2 and one more distal.
p53 alterations at the DNA, mRNA and protein levels were studied in tumour metastases sampled fro... more p53 alterations at the DNA, mRNA and protein levels were studied in tumour metastases sampled from 30 patients with malignant melanoma. Paraffin-embedded sections from these and an additional 12 patients were examined for the presence of p53 protein. TP53 gene aberrations were found in 7 of 30 (23%) of the patients, six of which showed loss of heterozygosity (LOH). Point mutations were detected in only two cases, one of which had LOH whereas the other was non-informative. Increased levels of p53 mRNA were present in only one tumour with, but in six cases without, detectable DNA abnormalities. Four of the latter and six tumours with normal transcript levels had immunohistochemically detectable levels of p53 protein. In 25 cases in which corresponding primary and metastatic lesions could be compared, closely similar immunoreactivity patterns were observed. Increased expression of the MDM2 gene was found in only one tumour in parallel with overexpression of p53. Altogether, the data indicate that inactivation of the p53 regulatory pathway is not of major significance in the tumorigenesis of malignant melanoma. However, a significant association was found between p53 immunoreactivity and the relapse-free period in patients with superficial spreading melanoma. That increased protein expression was predominantly found in tumours without DNA alterations might suggest a role for the wild-type p53 protein in restricting malignant cell proliferation in these cases.
In a European joint project carried out in 6 laboratories a disease-oriented program was set up c... more In a European joint project carried out in 6 laboratories a disease-oriented program was set up consisting of a panel of 7 tumor types, each represented by 4 to 8 different human tumor lines, for secondary screening of promising anticancer drugs. Human tumor lines were selected on the basis of differences in histology, growth rate, and sensitivity to conventional cytostatic agents. Xenografts were grown s.c. in nude mice, and treatment was started when tumors reached a mean diameter of 6 mm in groups of mice where at least 6 tumors were evaluable. Drugs were given at the maximum tolerated dose. For evaluation of drug efficacy, median tumor growth curves were drawn, and specific growth delay and treated/control x 100% were calculated. Doxorubicin (8 mg/kg i.v. days 1 and 8) was effective (treated/control < 50%, and specific growth delay > 1.0) in 0 of 2 breast cancers, 1 of 3 colorectal cancers, 2 of 5 head and neck cancers, 3 of 6 non-small cell lung cancers, 4 of 6 small cell...
Tumor cells and their surrounding microenvironment produce a variety of factors that promote tumo... more Tumor cells and their surrounding microenvironment produce a variety of factors that promote tumor growth and metastasis. We recently identified a nuclear factor, termed com1, that is up-regulated in human breast carcinoma cells on formation of experimental metastatic tumors and is assumed to act as a growth-promoting factor in breast cancer. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is a potent inhibitor of growth in breast cancer both in vitro and in vivo. We compared the growth-regulatory mechanisms of nontumorigenic and estrogen-dependent MCF-7 cells with those of the tumorigenic and tamoxifen-resistant subline MCF7/ LCC2 in the presence of 1,25(OH)2D3. Proliferation of MCF7/LCC2 cells, which revealed constitutive com1 expression, was inhibited by 1,25(OH)2D3 (10(-7) M). This was strongly associated with cell cycle arrest in G1 phase, consistent with accumulation of the hypophosphorylated form of the retinoblastoma protein as well as the induction of the cyclin-dependent kinase inh...
The antineoplastic efficacy of P-4055, a 5'-elaidic acid (C18:1, unsaturated fatty acid) este... more The antineoplastic efficacy of P-4055, a 5'-elaidic acid (C18:1, unsaturated fatty acid) ester of cytarabine, a nucleoside antimetabolite frequently used in the treatment of hematological malignancies, was examined in several in vivo models for human cancer. In initial dose-finding studies in nude mice, the efficacy of P-4055 was highest when using schedules with repeated daily doses. In a Raji Burkitt's lymphoma leptomeningeal carcinomatosis model in nude rats, the control cytarabine- and saline-treated animals (five in each group) had a mean survival time of 13.2 days, whereas treatment with P-4055 resulted in three of five long-time survivors (>70 days). In a systemic Raji leukemia model in nude mice, 8 of 10 of the P-4055-treated animals survived (>80 days), compared with none of the cytarabine-treated animals (mean survival time, 34.2 days). In s.c. xenograft models, the effects of maximum tolerated doses of P-4055 and cytarabine, given in four weekly cycles of da...
Clinical cancer research : an official journal of the American Association for Cancer Research, 1997
Tissue inhibitors of metalloproteinases (TIMPs) are believed to possess several cellular function... more Tissue inhibitors of metalloproteinases (TIMPs) are believed to possess several cellular functions, particularly the contrasting activities of inhibiting tissue-degrading enzymes and promoting cellular growth. In attempts to elucidate which of these functions may prevail in breast cancer, expression of mRNAs for TIMP-1 and TIMP-2 in the primary carcinomas from 34 breast cancer patients was related to known prognostic parameters and the clinical outcome. High levels of TIMP-1 mRNA showed significant correlation with the presence of lymph node metastases (P = 0.0067), development of distant metastases (P = 0.014), and early death of the disease (P = 0.020). Elevated expression of TIMP-2 mRNA was associated with development of distant metastases (P = 0.0055). No correlations, however, were observed between mRNA levels of TIMPs and prognostic factors such as patient age, tumor size, grade of anaplasia, or steroid receptor status; neither were any correlations found between these clinico...
The putative role of the CAPL gene in enhancing the development of human cancer metastasis was ex... more The putative role of the CAPL gene in enhancing the development of human cancer metastasis was examined by transfecting human high-expressing osteosarcoma cells with a hammerhead ribozyme directed against the gene transcript. The ability of the ribozyme to cleave target mRNA in intact cells was demonstrated in a 5'-rapid amplification of cDNA ends assay. In transfected cells, a suppression of the capacity to give skeletal metastases upon intracardial injection into nude rats was observed in cell clones with reduced expression of CAPL mRNA and protein, whereas in vitro and in vivo cell proliferation and tumorigenicity were unchanged. The results provide direct evidence that the expression level of the CAPL-encoded protein can determine the metastatic potential of osteosarcoma cells, and they demonstrate an association between reduced gene expression and proliferation-independent inhibition of the metastatic capacity of human tumor cells. The effects of the specific cleavage of CA...
Immunohistochemical analysis of the expression of the cyclin kinase inhibitor p21WAF1/CIP1 in a p... more Immunohistochemical analysis of the expression of the cyclin kinase inhibitor p21WAF1/CIP1 in a panel of primary and metastatic human melanocytic tumors was performed. It was found that, independent of the p53 status, approximately 30% of the primary melanomas and 40% of the metastases completely lacked expression of this cell cycle inhibitor. Some tumors were also analyzed by Northern blotting, and in most of the cases a consistant correlation between mRNA and protein expression was observed. In four benign nevi studied, WAF1/CIP1 mRNA was expressed whereas the protein was not detected, suggesting a post-transcriptional regulation of the inhibitor in these cases. In superficial spreading melanomas, a significant correlation between protein expression and tumor thickness was found, with thin lesions showing low protein levels. Interestingly, by comparing primary and metastatic specimens obtained from the same patient, a reduction in p21WAF1/CIP1 antibody staining was observed in the...
The potential of autologous bone marrow transplantation to improve the treatment results for pati... more The potential of autologous bone marrow transplantation to improve the treatment results for patients with small cell lung cancer (SCLC) may be limited by the presence of tumor cells in the graft. We constructed immunotoxins (ITs) involving 4 monoclonal antibodies and Pseudomonas exotoxin A and investigated the cytotoxicity of the ITs to H-146 SCLC cells in the presence and absence of normal human bone marrow (BM) cells. The Pseudomonas exotoxin A conjugate with the MOC-1 antibody, which recognizes an NCAM antigen, was inactive, as tested in a reproducible soft agar assay. Conjugates involving the monoclonal antibodies MOC-31, NrLu10, and MLuC1 killed about 3.5 log tumor cells at 0.1 microgram/ml and > 5.0 log at 1 microgram/ml. In the absence of BM cells, the combination of the 3 ITs was not superior to each IT used individually. However, when H-146 cells were admixed to nucleated BM cells at the ratio of 1:10, > 5 log tumor cell kill was obtained at a concentration as low as...
Intratibial injection in nude rats of 1 x 10(6) OHS, MHMX, and LOX human tumor cells resulted in ... more Intratibial injection in nude rats of 1 x 10(6) OHS, MHMX, and LOX human tumor cells resulted in each case in progressively growing bone tumors. When the diameter of the affected leg had increased by 2-3 mm, the animals were examined for uptake of 99mTc-methylenediphosphonate. The OHS osteosarcoma tumors caused sclerotic lesions with high and uniform isotope uptake, and the MHMX unclassified sarcoma showed a mixed pattern with both sclerotic and lytic areas, whereas the LOX melanoma caused lytic bone lesions with low uptake of the radionuclide. These findings were compared with the results of analogous investigations previously performed in the patients from whom the tumor lines originated. Striking similarities in both the morphology and the scintigraphic images were observed between corresponding tumors in rats and humans, with results supporting the clinical relevance of the model systems. When the LOX model was used for therapy experiments, doxorubicin had no effect on the growt...
Tissue from 19 human testis tumors was transplanted into athymic mice. One embryonal carcinoma, E... more Tissue from 19 human testis tumors was transplanted into athymic mice. One embryonal carcinoma, ECCS, grew rapidly, and this tumor was studied both as a xenograft and an in vitro culture of xenograft-derived tumor cells. Xenografts showed no evidence of differentiation. The embryonal carcinoma cells were heteroploid and showed alkaline phosphatase activity. When tumor cells from the xenografts were grown in vitro, the cells formed aggregates resembling embryoid bodies with epithelium-like cells in the periphery. Regularly, another population of mouse cells which showed several criteria of malignancy overgrew the culture and could be subcultured continuously. These abnormal cells may result from an in vivo or in vitro transformation of mouse stromal cells.
The growth of 6 different human melanomas xenografted into athymic mice was followed by measuring... more The growth of 6 different human melanomas xenografted into athymic mice was followed by measuring 2 vertical diameters twice weekly. The growth rate of the xenografts was expressed as the tumour doubling time, measured in the volume range 30-60 mm3. None of the xenografts increased their growth rate with increasing number of passages. The tumour lines, which were histologically very similar, showed individual, characteristic growth rates differing by a factor of 3. Evidence was obtained that resistance to cytostatic drugs in the patients is retained in the xenograft. Procedures for evaluating response to chemotherapy are discussed.
The chemosensitivity of human tumor xenografts to mitozolomide, 8-carbamoyl-3-(2-chloroethyl)imid... more The chemosensitivity of human tumor xenografts to mitozolomide, 8-carbamoyl-3-(2-chloroethyl)imidazo[5-1-d]-1,2,3,5-tetrazin-4(3H) -one, was studied in 3 different assay systems. In concentrations of 1 to 500 micrograms/ml, mitozolomide completely inhibited the colony-forming ability in soft agar of cell suspensions from sarcomas, melanomas, lung and colon cancers, and a mammary carcinoma. When a panel of tumors of the different histological types was tested for its sensitivity to mitozolomide in vitro, in the 6-day subrenal capsule assay in conventional mice, and, in some cases, as s.c. growing tumors in nude mice, good agreement between the different assay systems was seen. In most cases, a very pronounced antitumor effect was observed. The efficacy of mitozolomide was as good or better than that of the drugs clinically used against the tumor types tested. Tumor size measurements and histological examinations indicated that nude mice carrying a melanoma, a small cell lung cancer, ...
Ampli®ed segments of the long arm of chromosome 12 are frequently observed in human sarcomas. In ... more Ampli®ed segments of the long arm of chromosome 12 are frequently observed in human sarcomas. In most cases there are separate ampli®ed regions around the MDM2 and CDK4 genes. Here we show recurrent ampli®cation of a third region encompassing HMGIC, a human architectural transcription factor gene. Reduced ampli®cation frequency of sequences¯anking the gene was observed, indicating that inclusion of this third region in the amplicons is also selected for. In three samples only the 5' part of HMGIC was ampli®ed, suggesting preferential loss of the 3' part of the gene preceding or during ampli®cation. In several other samples rearrangement of the gene was observed. Expression analysis showed transcripts of aberrant sizes, lacking 3' sequences, and 3' RACE of one sample revealed replacement of exons 4 and 5 with ectopic sequences. This truncation of HMGIC resembles that reported for translocations of HMGIC in benign tumors, including lipomas, and it is striking that the gene was frequently ampli®ed or rearranged in well dierentiated liposarcomas, the malignant counterpart of lipomas. It seems conceivable that high levels of either full length or truncated hmgic could be relevant for the etiology of these tumors
Aim-The presence of malignant cells in the blood and bone marrow of patients with cancer at the t... more Aim-The presence of malignant cells in the blood and bone marrow of patients with cancer at the time of surgery may be indicative of early relapse. In addition to their numbers, the phenotypes of the micrometastatic cells might be essential in determining whether overt metastases will develop. This study aimed to establish a sensitive method for the detection and characterisation of malignant cells present in bone marrow. Methods-In spiking experiments, SKBR3 cells were mixed with mononuclear cells in known proportions to mimic bone marrow samples with micrometastatic cells. Tumour cells were extracted using SAM-M450 Dynabeads coupled to the MOC-31 anti-epithelial antibody, and were further analysed for amplification of erbB2 and int2 by fluorescent in situ hybridisation (FISH). erbB2 and int2 copy numbers were also determined in 15 primary breast cancers, and bone marrow samples from patients with amplification were analysed for micrometastatic cells by immunomagnetic enrichment and FISH. Results-In model experiments, cells with amplification could be detected in bead selected fractions when ratios of tumour cells (SKBR3) to mononuclear cells were as low as 10:10 7. Among the tumour samples, eight showed increased copy numbers of erbB2 and/or int2, and three of these patients had detectable numbers of tumour cells in their bone marrow: 4000, 540, and 26 tumour cells/10 7 mononuclear cells, respectively. The patient with 540 tumour cells/10 7 mononuclear cells showed high level amplification of erbB2 and suVered from a particularly aggressive disease, whereas the patient with 4000 tumour cells/10 7 mononuclear cells had favourable disease progression. Conclusion-These results demonstrate the feasibility and advantage of combining immunomagnetic selection and FISH characterisation of cancer cells in bone marrow samples. It is possible that molecular characterisation of such cells could provide prognostically valuable information.
F ig u re 1 Partial synovi al s arcoma-derived karyotype sh owing tb e normal chromosomes X and 1... more F ig u re 1 Partial synovi al s arcoma-derived karyotype sh owing tb e normal chromosomes X and 18 and its translocation derivatives X/ 18 and 18/X.
Despite recent technical improvements in surgical excision techniques and adjuvant radio-and chem... more Despite recent technical improvements in surgical excision techniques and adjuvant radio-and chemotherapy, the clinical outcome of patients with grade IV astrocytoma (glioblastoma) remains very poor, with a median survival of less than 12 months. A promising approach to therapy employs gene-engineered neural stem/progenitor cells (NSCs) as a cellular therapeutic delivery system, to track glioblastoma cells and deliver anticancer molecules. However, most results on their tumor tropism have been derived by immortalized NSCs. We now report that primary murine gene-engineered NSCs displayed in vivo tropism for glioblastoma cells. Ten days after injection into the brain, many NSCs continued to express the transgene and the NSC marker, nestin. NSCs transduced with a retroviral vector co-expressing a secretable form of human endostatin and eGFP (NSC/endo-eGFP) released potentially antiangiogenetic concentrations of endostatin into the culture medium. Conditioned medium of NSC/endo-eGFP cells inhibited the growth of mouse pulmonary microvascular endothelial cells (PMVECs). A good correlation between endostatin levels and PMVEC growth inhibition was observed. In NSCs co-expressing cytochrome P450 2B6 (CYP2B6) and eGFP (NSC/CYP2B6-eGFP), the forced expression of CYP2B6 resulted in intracellular activation of CPA and subsequent cell death. In the presence of NSC/CYP2B6-eGFP, we observed CPA cytotoxicity to DsRed-expressing U87Mg glioma cells. In vivo treatment of intracranial GL-261 glioblastoma with NSC/endo-eGFP caused a 65% reduction in tumor size, compared to untreated control mice or mice treated with NSC/eGFP cells. Our data suggest that primary NSCs transduced with retroviral vectors expressing endostatin and/or CYP2B6 have a potential role in glioblastoma therapy.
A phase II trial with mitozolomide was carried out in patients with malignant melanoma, since in ... more A phase II trial with mitozolomide was carried out in patients with malignant melanoma, since in preclinical studies this new imidazotetrazine had shown promising effects against human melanoma xenografts. Twenty-one evaluable patients with advanced malignant melanoma were teated with 115mgm-2 of mitozolomide, given orally every 6 weeks. None of the patients had received prior chemotherapy. Two partial responses (10 and 7+ months) were observed. The responding patients had lung metastases, and one of them had, in addition, a huge (17 x 14 cm) lymph node metastasis in the groin. Also, one patient had a 48% tumour volume reduction of lung metastases. The dose limiting side effect of the treatment was bone marrow depression, with delayed leukopenia and thrombocytopenia. The median white blood cell counts and platelet nadirs were 2.5 x 109 1-1 (range 1.1-3.8) and 59 x 109 11 (range 14-95), respectively. Nonhaematological adverse reactions were limited to mild or moderate nausea. It is concluded that orally administered mitozolomide is active against malignant melanoma and seems to have a response rate comparable to those of the most active established drugs.
In a recent comparative genomic hybridization (CGH) study of a panel of sarcomas, we detected rec... more In a recent comparative genomic hybridization (CGH) study of a panel of sarcomas, we detected recurrent amplification of 1q21-q22 in soft tissue and bone tumours. Amplification of this region had not previously been associated with sarcoma development, but occasional amplification of CACY/S100A6 and MUC1 in 1q21 had been reported for melanoma and breast carcinoma respectively. Initial screening by Southern blot analysis showed amplification of S100A6, FLG and SPRR3 in several sarcomas and, in a first attempt to characterize the 1q21-q22 amplicon in more detail, we have now investigated the amplification status of these and 11 other markers in the region in 35 sarcoma samples. FLG was the most frequently amplified gene, and the markers located in the same 4.5-Mb region as FLG showed a higher incidence of amplification than the more distal ones. However, for most of the 14 markers, amplification levels were low, and only APOA2 and the anonymous marker D1S3620 showed high-level amplifications (> tenfold increases) in one sample each. We used fluorescence in situ hybridization (FISH) to determine the amplification patterns of two overlapping yeast artificial chromosomes (YACs) covering the region between Dl S3620 and FLG (789f2 and 764a1), as well as two more distally located YACs in nine selected samples. Six samples had amplification of the YAC containing DlS3620 and, in three, 764a1 was also included. Five of these tumours showed normal copies of the more distal YACs; thus, it seems likely that an important gene may be located within 789f2, or very close. Two samples had high copy numbers of the most distal YACs. Taken together, FISH and molecular analyses indicate complex amplification patterns in 1q21-q22 with at least two amplicons: one located near Dl S3620/789f2 and one more distal.
p53 alterations at the DNA, mRNA and protein levels were studied in tumour metastases sampled fro... more p53 alterations at the DNA, mRNA and protein levels were studied in tumour metastases sampled from 30 patients with malignant melanoma. Paraffin-embedded sections from these and an additional 12 patients were examined for the presence of p53 protein. TP53 gene aberrations were found in 7 of 30 (23%) of the patients, six of which showed loss of heterozygosity (LOH). Point mutations were detected in only two cases, one of which had LOH whereas the other was non-informative. Increased levels of p53 mRNA were present in only one tumour with, but in six cases without, detectable DNA abnormalities. Four of the latter and six tumours with normal transcript levels had immunohistochemically detectable levels of p53 protein. In 25 cases in which corresponding primary and metastatic lesions could be compared, closely similar immunoreactivity patterns were observed. Increased expression of the MDM2 gene was found in only one tumour in parallel with overexpression of p53. Altogether, the data indicate that inactivation of the p53 regulatory pathway is not of major significance in the tumorigenesis of malignant melanoma. However, a significant association was found between p53 immunoreactivity and the relapse-free period in patients with superficial spreading melanoma. That increased protein expression was predominantly found in tumours without DNA alterations might suggest a role for the wild-type p53 protein in restricting malignant cell proliferation in these cases.
In a European joint project carried out in 6 laboratories a disease-oriented program was set up c... more In a European joint project carried out in 6 laboratories a disease-oriented program was set up consisting of a panel of 7 tumor types, each represented by 4 to 8 different human tumor lines, for secondary screening of promising anticancer drugs. Human tumor lines were selected on the basis of differences in histology, growth rate, and sensitivity to conventional cytostatic agents. Xenografts were grown s.c. in nude mice, and treatment was started when tumors reached a mean diameter of 6 mm in groups of mice where at least 6 tumors were evaluable. Drugs were given at the maximum tolerated dose. For evaluation of drug efficacy, median tumor growth curves were drawn, and specific growth delay and treated/control x 100% were calculated. Doxorubicin (8 mg/kg i.v. days 1 and 8) was effective (treated/control < 50%, and specific growth delay > 1.0) in 0 of 2 breast cancers, 1 of 3 colorectal cancers, 2 of 5 head and neck cancers, 3 of 6 non-small cell lung cancers, 4 of 6 small cell...
Tumor cells and their surrounding microenvironment produce a variety of factors that promote tumo... more Tumor cells and their surrounding microenvironment produce a variety of factors that promote tumor growth and metastasis. We recently identified a nuclear factor, termed com1, that is up-regulated in human breast carcinoma cells on formation of experimental metastatic tumors and is assumed to act as a growth-promoting factor in breast cancer. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is a potent inhibitor of growth in breast cancer both in vitro and in vivo. We compared the growth-regulatory mechanisms of nontumorigenic and estrogen-dependent MCF-7 cells with those of the tumorigenic and tamoxifen-resistant subline MCF7/ LCC2 in the presence of 1,25(OH)2D3. Proliferation of MCF7/LCC2 cells, which revealed constitutive com1 expression, was inhibited by 1,25(OH)2D3 (10(-7) M). This was strongly associated with cell cycle arrest in G1 phase, consistent with accumulation of the hypophosphorylated form of the retinoblastoma protein as well as the induction of the cyclin-dependent kinase inh...
The antineoplastic efficacy of P-4055, a 5'-elaidic acid (C18:1, unsaturated fatty acid) este... more The antineoplastic efficacy of P-4055, a 5'-elaidic acid (C18:1, unsaturated fatty acid) ester of cytarabine, a nucleoside antimetabolite frequently used in the treatment of hematological malignancies, was examined in several in vivo models for human cancer. In initial dose-finding studies in nude mice, the efficacy of P-4055 was highest when using schedules with repeated daily doses. In a Raji Burkitt's lymphoma leptomeningeal carcinomatosis model in nude rats, the control cytarabine- and saline-treated animals (five in each group) had a mean survival time of 13.2 days, whereas treatment with P-4055 resulted in three of five long-time survivors (>70 days). In a systemic Raji leukemia model in nude mice, 8 of 10 of the P-4055-treated animals survived (>80 days), compared with none of the cytarabine-treated animals (mean survival time, 34.2 days). In s.c. xenograft models, the effects of maximum tolerated doses of P-4055 and cytarabine, given in four weekly cycles of da...
Clinical cancer research : an official journal of the American Association for Cancer Research, 1997
Tissue inhibitors of metalloproteinases (TIMPs) are believed to possess several cellular function... more Tissue inhibitors of metalloproteinases (TIMPs) are believed to possess several cellular functions, particularly the contrasting activities of inhibiting tissue-degrading enzymes and promoting cellular growth. In attempts to elucidate which of these functions may prevail in breast cancer, expression of mRNAs for TIMP-1 and TIMP-2 in the primary carcinomas from 34 breast cancer patients was related to known prognostic parameters and the clinical outcome. High levels of TIMP-1 mRNA showed significant correlation with the presence of lymph node metastases (P = 0.0067), development of distant metastases (P = 0.014), and early death of the disease (P = 0.020). Elevated expression of TIMP-2 mRNA was associated with development of distant metastases (P = 0.0055). No correlations, however, were observed between mRNA levels of TIMPs and prognostic factors such as patient age, tumor size, grade of anaplasia, or steroid receptor status; neither were any correlations found between these clinico...
The putative role of the CAPL gene in enhancing the development of human cancer metastasis was ex... more The putative role of the CAPL gene in enhancing the development of human cancer metastasis was examined by transfecting human high-expressing osteosarcoma cells with a hammerhead ribozyme directed against the gene transcript. The ability of the ribozyme to cleave target mRNA in intact cells was demonstrated in a 5'-rapid amplification of cDNA ends assay. In transfected cells, a suppression of the capacity to give skeletal metastases upon intracardial injection into nude rats was observed in cell clones with reduced expression of CAPL mRNA and protein, whereas in vitro and in vivo cell proliferation and tumorigenicity were unchanged. The results provide direct evidence that the expression level of the CAPL-encoded protein can determine the metastatic potential of osteosarcoma cells, and they demonstrate an association between reduced gene expression and proliferation-independent inhibition of the metastatic capacity of human tumor cells. The effects of the specific cleavage of CA...
Immunohistochemical analysis of the expression of the cyclin kinase inhibitor p21WAF1/CIP1 in a p... more Immunohistochemical analysis of the expression of the cyclin kinase inhibitor p21WAF1/CIP1 in a panel of primary and metastatic human melanocytic tumors was performed. It was found that, independent of the p53 status, approximately 30% of the primary melanomas and 40% of the metastases completely lacked expression of this cell cycle inhibitor. Some tumors were also analyzed by Northern blotting, and in most of the cases a consistant correlation between mRNA and protein expression was observed. In four benign nevi studied, WAF1/CIP1 mRNA was expressed whereas the protein was not detected, suggesting a post-transcriptional regulation of the inhibitor in these cases. In superficial spreading melanomas, a significant correlation between protein expression and tumor thickness was found, with thin lesions showing low protein levels. Interestingly, by comparing primary and metastatic specimens obtained from the same patient, a reduction in p21WAF1/CIP1 antibody staining was observed in the...
The potential of autologous bone marrow transplantation to improve the treatment results for pati... more The potential of autologous bone marrow transplantation to improve the treatment results for patients with small cell lung cancer (SCLC) may be limited by the presence of tumor cells in the graft. We constructed immunotoxins (ITs) involving 4 monoclonal antibodies and Pseudomonas exotoxin A and investigated the cytotoxicity of the ITs to H-146 SCLC cells in the presence and absence of normal human bone marrow (BM) cells. The Pseudomonas exotoxin A conjugate with the MOC-1 antibody, which recognizes an NCAM antigen, was inactive, as tested in a reproducible soft agar assay. Conjugates involving the monoclonal antibodies MOC-31, NrLu10, and MLuC1 killed about 3.5 log tumor cells at 0.1 microgram/ml and > 5.0 log at 1 microgram/ml. In the absence of BM cells, the combination of the 3 ITs was not superior to each IT used individually. However, when H-146 cells were admixed to nucleated BM cells at the ratio of 1:10, > 5 log tumor cell kill was obtained at a concentration as low as...
Intratibial injection in nude rats of 1 x 10(6) OHS, MHMX, and LOX human tumor cells resulted in ... more Intratibial injection in nude rats of 1 x 10(6) OHS, MHMX, and LOX human tumor cells resulted in each case in progressively growing bone tumors. When the diameter of the affected leg had increased by 2-3 mm, the animals were examined for uptake of 99mTc-methylenediphosphonate. The OHS osteosarcoma tumors caused sclerotic lesions with high and uniform isotope uptake, and the MHMX unclassified sarcoma showed a mixed pattern with both sclerotic and lytic areas, whereas the LOX melanoma caused lytic bone lesions with low uptake of the radionuclide. These findings were compared with the results of analogous investigations previously performed in the patients from whom the tumor lines originated. Striking similarities in both the morphology and the scintigraphic images were observed between corresponding tumors in rats and humans, with results supporting the clinical relevance of the model systems. When the LOX model was used for therapy experiments, doxorubicin had no effect on the growt...
Tissue from 19 human testis tumors was transplanted into athymic mice. One embryonal carcinoma, E... more Tissue from 19 human testis tumors was transplanted into athymic mice. One embryonal carcinoma, ECCS, grew rapidly, and this tumor was studied both as a xenograft and an in vitro culture of xenograft-derived tumor cells. Xenografts showed no evidence of differentiation. The embryonal carcinoma cells were heteroploid and showed alkaline phosphatase activity. When tumor cells from the xenografts were grown in vitro, the cells formed aggregates resembling embryoid bodies with epithelium-like cells in the periphery. Regularly, another population of mouse cells which showed several criteria of malignancy overgrew the culture and could be subcultured continuously. These abnormal cells may result from an in vivo or in vitro transformation of mouse stromal cells.
The growth of 6 different human melanomas xenografted into athymic mice was followed by measuring... more The growth of 6 different human melanomas xenografted into athymic mice was followed by measuring 2 vertical diameters twice weekly. The growth rate of the xenografts was expressed as the tumour doubling time, measured in the volume range 30-60 mm3. None of the xenografts increased their growth rate with increasing number of passages. The tumour lines, which were histologically very similar, showed individual, characteristic growth rates differing by a factor of 3. Evidence was obtained that resistance to cytostatic drugs in the patients is retained in the xenograft. Procedures for evaluating response to chemotherapy are discussed.
The chemosensitivity of human tumor xenografts to mitozolomide, 8-carbamoyl-3-(2-chloroethyl)imid... more The chemosensitivity of human tumor xenografts to mitozolomide, 8-carbamoyl-3-(2-chloroethyl)imidazo[5-1-d]-1,2,3,5-tetrazin-4(3H) -one, was studied in 3 different assay systems. In concentrations of 1 to 500 micrograms/ml, mitozolomide completely inhibited the colony-forming ability in soft agar of cell suspensions from sarcomas, melanomas, lung and colon cancers, and a mammary carcinoma. When a panel of tumors of the different histological types was tested for its sensitivity to mitozolomide in vitro, in the 6-day subrenal capsule assay in conventional mice, and, in some cases, as s.c. growing tumors in nude mice, good agreement between the different assay systems was seen. In most cases, a very pronounced antitumor effect was observed. The efficacy of mitozolomide was as good or better than that of the drugs clinically used against the tumor types tested. Tumor size measurements and histological examinations indicated that nude mice carrying a melanoma, a small cell lung cancer, ...
Ampli®ed segments of the long arm of chromosome 12 are frequently observed in human sarcomas. In ... more Ampli®ed segments of the long arm of chromosome 12 are frequently observed in human sarcomas. In most cases there are separate ampli®ed regions around the MDM2 and CDK4 genes. Here we show recurrent ampli®cation of a third region encompassing HMGIC, a human architectural transcription factor gene. Reduced ampli®cation frequency of sequences¯anking the gene was observed, indicating that inclusion of this third region in the amplicons is also selected for. In three samples only the 5' part of HMGIC was ampli®ed, suggesting preferential loss of the 3' part of the gene preceding or during ampli®cation. In several other samples rearrangement of the gene was observed. Expression analysis showed transcripts of aberrant sizes, lacking 3' sequences, and 3' RACE of one sample revealed replacement of exons 4 and 5 with ectopic sequences. This truncation of HMGIC resembles that reported for translocations of HMGIC in benign tumors, including lipomas, and it is striking that the gene was frequently ampli®ed or rearranged in well dierentiated liposarcomas, the malignant counterpart of lipomas. It seems conceivable that high levels of either full length or truncated hmgic could be relevant for the etiology of these tumors
Aim-The presence of malignant cells in the blood and bone marrow of patients with cancer at the t... more Aim-The presence of malignant cells in the blood and bone marrow of patients with cancer at the time of surgery may be indicative of early relapse. In addition to their numbers, the phenotypes of the micrometastatic cells might be essential in determining whether overt metastases will develop. This study aimed to establish a sensitive method for the detection and characterisation of malignant cells present in bone marrow. Methods-In spiking experiments, SKBR3 cells were mixed with mononuclear cells in known proportions to mimic bone marrow samples with micrometastatic cells. Tumour cells were extracted using SAM-M450 Dynabeads coupled to the MOC-31 anti-epithelial antibody, and were further analysed for amplification of erbB2 and int2 by fluorescent in situ hybridisation (FISH). erbB2 and int2 copy numbers were also determined in 15 primary breast cancers, and bone marrow samples from patients with amplification were analysed for micrometastatic cells by immunomagnetic enrichment and FISH. Results-In model experiments, cells with amplification could be detected in bead selected fractions when ratios of tumour cells (SKBR3) to mononuclear cells were as low as 10:10 7. Among the tumour samples, eight showed increased copy numbers of erbB2 and/or int2, and three of these patients had detectable numbers of tumour cells in their bone marrow: 4000, 540, and 26 tumour cells/10 7 mononuclear cells, respectively. The patient with 540 tumour cells/10 7 mononuclear cells showed high level amplification of erbB2 and suVered from a particularly aggressive disease, whereas the patient with 4000 tumour cells/10 7 mononuclear cells had favourable disease progression. Conclusion-These results demonstrate the feasibility and advantage of combining immunomagnetic selection and FISH characterisation of cancer cells in bone marrow samples. It is possible that molecular characterisation of such cells could provide prognostically valuable information.
F ig u re 1 Partial synovi al s arcoma-derived karyotype sh owing tb e normal chromosomes X and 1... more F ig u re 1 Partial synovi al s arcoma-derived karyotype sh owing tb e normal chromosomes X and 18 and its translocation derivatives X/ 18 and 18/X.
Despite recent technical improvements in surgical excision techniques and adjuvant radio-and chem... more Despite recent technical improvements in surgical excision techniques and adjuvant radio-and chemotherapy, the clinical outcome of patients with grade IV astrocytoma (glioblastoma) remains very poor, with a median survival of less than 12 months. A promising approach to therapy employs gene-engineered neural stem/progenitor cells (NSCs) as a cellular therapeutic delivery system, to track glioblastoma cells and deliver anticancer molecules. However, most results on their tumor tropism have been derived by immortalized NSCs. We now report that primary murine gene-engineered NSCs displayed in vivo tropism for glioblastoma cells. Ten days after injection into the brain, many NSCs continued to express the transgene and the NSC marker, nestin. NSCs transduced with a retroviral vector co-expressing a secretable form of human endostatin and eGFP (NSC/endo-eGFP) released potentially antiangiogenetic concentrations of endostatin into the culture medium. Conditioned medium of NSC/endo-eGFP cells inhibited the growth of mouse pulmonary microvascular endothelial cells (PMVECs). A good correlation between endostatin levels and PMVEC growth inhibition was observed. In NSCs co-expressing cytochrome P450 2B6 (CYP2B6) and eGFP (NSC/CYP2B6-eGFP), the forced expression of CYP2B6 resulted in intracellular activation of CPA and subsequent cell death. In the presence of NSC/CYP2B6-eGFP, we observed CPA cytotoxicity to DsRed-expressing U87Mg glioma cells. In vivo treatment of intracranial GL-261 glioblastoma with NSC/endo-eGFP caused a 65% reduction in tumor size, compared to untreated control mice or mice treated with NSC/eGFP cells. Our data suggest that primary NSCs transduced with retroviral vectors expressing endostatin and/or CYP2B6 have a potential role in glioblastoma therapy.
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Papers by O. Fodstad