The human genome puzzle - the role of copy number variation in somatic mosaicism.

The discovery of copy number variations (CNV) in the human genome opened new perspectives in the study of the genetic causes of inherited disorders and the etiology of common diseases. Differently patterned instances of somatic mosaicism in CNV regions have been shown to be present in monozygotic twins and throughout different tissues within an individual. A single-cell-level investigation of CNV in different human cell types led us to uncover mitotically derived genomic mosaicism, which is stable in different cell types of one individual. A unique study of immortalized B-lymphoblastoid cell lines obtained with 20 year interval from the same two subjects shows that mitotic changes in CNV regions may happen early during embryonic development and seem to occur only once, as levels of mosaicism remained stable. This finding has the potential to change our concept of dynamic human genome variation. We propose that further genomic studies should focus on the single-cell level, to understand better the etiology and physiology of aging and diseases mediated by somatic variations.

Diagnosis and therapy of oral squamous cell carcinoma.

Oral squamous cell carcinoma ranks among the top ten most common cancers worldwide. Despite the success in diagnosis and therapy during the past 30 years, oral squamous cell carcinoma still belongs to the tumor types with a very unfavorable prognosis. In an effort to identify genomic alterations with prognostic relevance, we applied the comparative genomic hybridization technique on oral squamous cell carcinoma. The tumors exhibited from five up to 47 DNA copy number alterations, indicating a considerable degree of genomic imbalance. Out of 35 tumors, 19 showed a gain of chromosome band 7p12. Genomic imbalances were investigated by hierarchical cluster analysis and clustered image mapping to investigate whether genomic profiles correlate with clinical data. Results of the present investigation show that profiling of genomic imbalances in general, and especially of the epidermal growth factor receptor (EGFR) on 7p12, may be suitable as prognostic factors. In order to identify small-molecule inhibitors for EGFR, we established a database of 531 natural compounds derived from medicinal plants used in traditional Chinese medicine. Candidate compounds were identified by correlation analysis using the Kendall tau-test of IC50 values of tumor cell lines and microarray-based EGFR mRNA expression. Further validation was performed by molecular docking studies using the AutoDock program with the crystal structure of EGFR tyrosine kinase domain as docking template. We estimate these results will be a further step toward the ultimate goal of individualized, patient-adapted tumor treatment based on tumor molecular profiling.

Double minutes, cytogenetic equivalents of gene amplification, in human neoplasia - a review.

Double minutes are tiny spherical chromatin bodies of a few mega-base pairs of size which are found occasionally in hematopoietic neoplasia and more or less often in human solid tumors. They have been associated with worse prognosis and poor outcome of the malignancies where present. With the beginning era of molecular cytogenetics they could be defined as cytogenetic equivalents of amplified DNA sequences. The identification of involved chromosomal segments and their molecular nature led to the development of molecular genetic techniques for a rapid and reliable detection of prognostically important oncogene amplifications in human tumors and,as a consequence, to gene-targeted therapy.

Double minutes, cytogenetic equivalents of gene amplification, in human neoplasia ­ a review

Double minutes are tiny spherical chromatin bodies of a few mega-base pairs of size which are found occasionally in hematopoietic neoplasia and more or less often in human solid tumors. They have been associated with worse prognosis and poor outcome of the malignancies where present. With the beginning era of molecular cytogenetics they could be defined as cytogenetic equivalents of amplified DNA sequences. The identification of involved chromosomal segments and their molecular nature led to the development of molecular genetic techniques for a rapid and reliable detection of prognostically important oncogene amplifications in human tumors and, as a consequence, to gene-targeted therapy

Severely incapacitating mutations in patients with extreme short stature identify RNA-processing endoribonuclease RMRP as an essential cell growth regulator.

The growth of an individual is deeply influenced by the regulation of cell growth and division, both of which also contribute to a wide variety of pathological conditions, including cancer, diabetes, and inflammation. To identify a major regulator of human growth, we performed positional cloning in an autosomal recessive type of profound short stature, anauxetic dysplasia. Homozygosity mapping led to the identification of novel mutations in the RMRP gene, which was previously known to cause two milder types of short stature with susceptibility to cancer, cartilage hair hypoplasia, and metaphyseal dysplasia without hypotrichosis. We show that different RMRP gene mutations lead to decreased cell growth by impairing ribosomal assembly and by altering cyclin-dependent cell cycle regulation. Clinical heterogeneity is explained by a correlation between the level and type of functional impairment in vitro and the severity of short stature or predisposition to cancer. Whereas the cartilage hair hypoplasia founder mutation affects both pathways intermediately, anauxetic dysplasia mutations do not affect B-cyclin messenger RNA (mRNA) levels but do severely incapacitate ribosomal assembly via defective endonucleolytic cleavage. Anauxetic dysplasia mutations thus lead to poor processing of ribosomal RNA while allowing normal mRNA processing and, therefore, genetically separate the different functions of RNase MRP.

Genomic imbalances in human leukemia and lymphoma detected by comparative genomic hybridization (Review).

A large number and variety of neoplasias of the hematopoietic system have been successfully subjected to CGH analysis. The obtained data shed light on genomic alterations beyond the basic rearrangements known as 'causative aberrations' in many of these diseases. Some of these alterations seem to play an important role in disease progression and specificity of the disease. They can also be associated with clinical parameters like response to therapy and survival. The patterns of genomic alterations found by CGH can characterize certain disease entities and differentiate them from others. If the chromosomal segments affected in > 10% of the cases of each basic disease entity [acute myeloblastic leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and multiple myeloma (MM)] are compared, most of the frequently involved chromosomal regions differ from entity to entity in the leukemias. The only exception are losses on 13q which are common to CLL and multiple myeloma. However, these patterns can also deduce chromosomal locations basically involved in the processes of hematopoietic oncogenesis, which is particularly evident in lymphomas. For instance, gain of 18q is shared by all lymphoma entities presented, and gain of 3q, 7q and 12q is commonly found in three of the differentiated classes. It is also of practical interest to control the differences and consistencies of imbalances found in nodular and in organ-confined lymphomas. Besides aneuploidies, which can also be readily detected by chromosome banding, CGH defines imbalances of chromosomal segments, which can become the basis for searching for neoplasia-related genes. With respect to their clinical significance, the presence of genomic imbalances is associated with disease progression and, therefore, poorer prognosis.

Detection of cryptic chromosomal aberrations in the in vitro non-proliferating cells of acute myeloid leukemia.

A total of 22 acute myeloid leukemia (AML) cases were analyzed by cell-specific comparative genomic hybridization (micro-CGH). Conventional banding analysis identified a monosomy 7 in six (group I), a trisomy 8 in eight (group II) and a normal karyotype in eight cases (group III). A total of 32 additional chromosomal imbalances was detected and confirmed in two independent micro-CGH experiments. However, only in 9 of the 22 cases (group I: 4 cases; group II: 1 case; group III: 4 cases) the existence of 11 of the 32 (34.5%) detected copy number alterations could be confirmed by other fluorescence in situ hybridization (FISH) approaches. These results lead to two conclusions: i) in the in vitro non-proliferating population of AML tumor cells one can detect cryptic chromosomal aberrations, which might constitute tumor markers of diagnostic and prognostic value; ii) The results of CGH need to be checked by other approaches.

Fragility of telomeres after bleomycin and cisplatin combined treatment measured in human leukocytes with the Comet-FISH technique.

UNLABELLED: THE AIM of the present study was the comparative investigation of action of widely applied anticancer preparations: cisplatin (cis-DDP) and bleomycin (BLM) on total DNA and telomeres damage in human blood cells. METHODS: The "Comet-FISH technique" -- single cell gel electrophoresis ("comet assay") in combination with fluorescent in situ hybridization (FISH) was used for this purpose. This newly applied combined approach permits to detect on the same specimen the total DNA damage in individual cells and evaluate specific DNA sequences as well. Telomere -- specific -- PNA (peptide nucleic acid) probes were used for the localization of telomeres in the comet's head and their migration to the tail. THE RESULTS obtained indicate that in control variants, due to DNA metabolism and handling, approximately 7% of the DNA and 17% of the telomeres were found in the tail. In cells treated with BLM alone, telomeres leak out with equal probability as total DNA. In turn, the combination of cis-DDP with BLM reduces telomere migration more than the migration of total DNA due to cis-DDP crosslinking effect. Thus, preferentially telomeric action of the cis-DDP can be concluded. CONCLUSION: The Comet-FISH approach permitted us to reveal the induction of DNA breaks with BLM and its modification due to platinum-crosslink formation, using telomeric PNA probes.

Genomic gain of the epidermal growth factor receptor harboring band 7p12 is part of a complex pattern of genomic imbalances in oral squamous cell carcinomas.

BACKGROUND: To determine the association between changes of genomic gene dose and clinical parameters in squamous cell carcinomas of the oral cavity, comparative genomic hybridization seemed suited not only to detect genomic imbalances in these tumors, but also particularly to examine the role of gain of 7p12, the band harboring the epidermal growth factor receptor (EGFR) in this context. METHODS: Total genomic DNA obtained from 35 oral squamous cell carcinomas was subjected to comparative genomic hybridization (CGH) and detected patterns of genomic imbalances were associated with various clinical parameters. RESULTS: The examined tumors exhibited five and up to 47 DNA copy number alterations (CNAs). Nineteen of these showed a gain of chromosome band 7p12. A highly complex but strikingly consistent pattern of genomic imbalances (average, 32 CNAs per tumor) was associated with this alteration, among which gains clearly dominated over losses of genomic material. Comparable patterns, however, could also be found in a few tumors with a high number of CNAs (average, 26) but without the 7p gain. Low numbers of imbalances always were accompanied by low consistency of CNA patterns and none of these cases showed enh(7p12). No significant differences with respect to pT class or grade of tumors were found between enh(7p)-positive and -negative tumors. Stage IV and lymph node affection were slightly more frequent among enh(7p12)-positive than in -negative cases. Relapse occurred in 63% in 7p12-positive vs. 25% in the negative group. Average disease-free survival of tumors without 7p gain clearly exceeded that of tumors with gain of 7p (36.8 vs. 21.3). However, some of these associations could also be found if comparison was based on number of CNAs. By means of hierarchical cluster analysis, we were able to show that different patterns of CNAs can be separated from each other in tumors with or without 7p alterations, and that these patterns predict short- or long-term survival of patients. CONCLUSIONS: Previously described associations of gains of 7p12, the chromosomal band harboring the EGFR gene with clinical parameters can reasonably be estimated only within the context of the pattern and complexity of the genomic imbalances accompanying this chromosomal loss in examined tumors.

Comet-FISH using peptide nucleic acid probes detects telomeric repeats in DNA damaged by bleomycin and mitomycin C proportional to general DNA damage.

For the optimal use of anticancer drugs a knowledge of the whole spectrum of side-effects is required. A potential hazard, so far only scarcely investigated, is uncontrolled effects of drugs such as bleomycin (BLM) and mitomycin C (MMC) on telomere shortening in non-cancerous tissues of the treated person. For the first time, directly labelled telomere-specific peptide nucleic acid (PNA) hybridization probes were applied in comet-FISH to detect DNA fragmentation on an intermediate scale. The effects of BLM and MMC were measured in peripheral blood cells of three human volunteers, following ex vivo incubation. Fragmentation of telomeres and subtelomeric regions was highly specifically detected by the comet-FISH assay, a combination of the comet assay and fluorescence in situ hybridization. As a technical detail, the effects of the hybridization procedure have been studied on the level of single comets. Image analysis before and after the hybridization process reveals a small decrease in the detected fragmented DNA, probably due to diffusion of small fragments. It could not only be shown that both drugs actually induce breaks in telomere-associated DNA, but also that the comet-FISH technique, as a quantitative approach, is a useful tool for the detection and evaluation of the role of sequence-specific DNA damage after mutagenic action. The breakage frequency for DNA of or adjacent to telomeric repeats was found to be proportional to that of the total DNA, which hints at random induction of DNA breaks by BLM and MMC. In terms of therapy, the results indicate that no over- or under-proportional effects on telomeres of BLM or MMC need be expected.

Combination treatment of glioblastoma multiforme cell lines with the anti-malarial artesunate and the epidermal growth factor receptor tyrosine kinase inhibitor OSI-774.

New drugs and combination modalities for otherwise non-responsive brain tumors are urgently required. The anti-malarial artesunate (ART) and the EGFR tyrosine kinase inhibitor OSI-774 reveal profound cytotoxic activity. The effectiveness of a combination treatment and the underlying molecular determinants of cellular response are unknown. In the present investigation, we studied ART and OSI-774 in glioblastoma multiforme (GBM) cell lines. Supra-additive inhibition of cell growth was observed in U-87MG.DeltaEGFR cells transduced with a deletion-mutant constitutively active EGFR gene, while additive effects were present in cells transduced with wild-type EGFR (U-87MG.WT-2N), kinase-deficient EGFR (U-87MG.DK-2N), mock vector controls (U-87MG.LUX), or non-transduced parental U-87MG cells. Among nine other non-transduced GBM cell lines, supra-additive effects were found in two cell lines (G-210GM, G-599GM), while ART and OSI-774 acted in an additive manner in the other seven cell lines (G-211GM, G-750GM, G-1163GM, G-1187GM, G-1265GM, G-1301GM, and G-1408GM). Sub-additive or antagonistic effects were not observed. Genomic gains and losses of genetic material in the non-transduced cell lines as assessed by comparative genomic hybridization were correlated with the IC(50) values for ART and OSI-774 and subsequently subjected to hierarchical cluster analysis and cluster image mapping. A genomic profile of imbalances was detected that predicted cellular response to ART and OSI-774. The genes located at the genomic imbalances of interest may serve as candidate resistance genes of GBM cells towards ART and OSI-774. In conclusion, the combination treatment of ART and OSI-774 resulted in an increased growth inhibition of GBM cell lines as compared to each drug alone.

Inverse correlation of epidermal growth factor receptor messenger RNA induction and suppression of anchorage-independent growth by OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in glioblastoma multiforme cell lines.

OBJECT: Quantitative and qualitative alterations in the epidermal growth factor receptor (EGFR) commonly occur in many cancers in humans, including malignant gliomas. The aim of the current study was to evaluate molecular and cellular effects of OSI-774, a novel EGFR tyrosine kinase inhibitor, on nine glioblastoma multiforme (GBM) cell lines. METHODS: The effects of OSI-774 on expression of EGFR messenger (m)RNA and protein, proliferation, anchorage-independent growth, and apoptosis were examined using semiquantitative reverse transcription-polymerase chain reaction, immunocytochemical analysis, Coulter counting, soft agar cloning, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling/fluorescence-activated cell sorting, respectively. All p53 genes were completely and bidirectionally sequenced. Suppression of anchorage-independent growth by OSI-774 was inversely correlated to the induction of EGFR mRNA during relative serum starvation (r = -0.74) and was unrelated to p53 status. Overall, suppression of anchorage-independent growth was a considerably stronger effect of OSI-774 than inhibition of proliferation. The extent of OSI-774-induced apoptosis positively correlated with both proliferation and anchorage-independent growth of GBM cell lines (r = 0.75 and 0.79, respectively). In a single cell line derived from a secondary GBM, exposure to concentrations of greater than or equal to 1 micromol/L resulted in a substantial net cell loss during proliferation studies. CONCLUSIONS: The induction of EGFR mRNA may constitute a cellular mechanism to counteract the inhibitory effect of OSI-774 on the anchorage-independent growth of GBM cells. In contrast, no considerable correlation could be established between baseline expression levels of EGFR (both mRNA and protein) in GBM cell lines and their biological response to OSI-774. The OSI-774 induced greater (p53-independent) apoptosis in more malignant GBM phenotypes and may be a promising therapeutic agent against secondary GBM.

Breakpoint differentiation in chromosomal aberrations of hematological malignancies: Identification of 33 previously unrecorded breakpoints.

Routine cytogenetic analysis provides important information of diagnostic and prognostic relevance for hematological malignancies. In spite of this, poorly spread metaphase chromosomes and highly rearranged karyotypes with numerous marker chromosomes, are often difficult to interpret. In order to improve the definition of chromosomal breakpoints multicolor banding (MCB) was applied on 45 bone marrow samples from patients suffering from hematological malignancies like myelodysplastic syndrome (MDS), acute myelocytic leukemia (AML), chronic myelocytic leukemia (CML) or acute lymphoblastic leukemia (ALL). The breakpoints defined by GTG banding were confirmed by MCB in 8 cases, while in the remaining 37 cases the breakpoints had to be redefined. In 20/45 cases the breakpoints could only be characterized after application of MCB. In summary, 73 different breakpoints were characterized, thereof 33 were previously undescribed. Eleven cases showed known acquired aberrations and 21 cases had previously described aberration types such as del(5q-), del(7q-), del(13q-) or t(1;5) as sole rearrangement or in connection with other complex ones. In a total of 11 cases 19 breakpoints as described before were involved in hematological malignancies, while in 14 cases 33 breakpoints were identified which have not been described previously. Thus, MCB has proven to be a powerful and reliable method for screening of chromosomal aberrations, which considerably increased the accuracy of cytogenetic diagnosis.

Identification of gene expression profiles predicting tumor cell response to L-alanosine.

The methylthioadenosine phosphorylase (MTAP) gene gained considerable interest as therapeutic target for tumors with the 9p21 deletion. This gene maps to 9p21 and loss of this chromosomal region in tumors offers an unique opportunity for chemoselective treatment, since MTAP is an important salvage enzyme for the formation of adenine that is needed for DNA synthesis. L-Alanosine, an antibiotic from Streptomyces alanosinicus, blocks the common de novo purine biosynthesis pathway and, thereby, inhibits tumor cells with MTAP deficiency. Normal cells escape the detrimental effects of L-alanosine due to their proficiency in the MTAP salvage pathway. The present analysis was undertaken to gain insights into the molecular architecture of tumor cells that determines the response to L-alanosine apart from the MTAP gene. Analysis of cell doubling times and IC(50) values for L-alanosine showed that slowly growing cell lines were more resistant to L-alanosine than rapidly growing ones. Mining the database of the National Cancer Institute (N.C.I.), for the mRNA expression of 9706 genes in 60 cell lines by means of Kendall's tau-test, false discovery rate calculation, and hierarchical cluster analysis pointed to 11 genes or expressed sequence tags whose mRNA expression correlated with the IC(50) values for L-alanosine. Furthermore, we tested L-alanosine for cross-resistance in multidrug-resistant cell lines which overexpress selectively either the P-glycoprotein/MDR1 (CEM/ADR5000), MRP1 (HL-60/AR), or BCRP (MDA-MB-231-BCRP) genes. None of the multidrug-resistant cell lines was cross-resistant to L-alanosine indicating that L-alanosine may be suitable to treat multidrug-resistant, refractory tumors in the clinic. Finally, the IC(50) values for L-alanosine of the 60 cell lines were correlated to the p53 mutational status and expression of p53 downstream genes. We found that p53 mutated cell lines were more resistant to L-alanosine than p53 wild type cell lines.

Molecular modes of action of artesunate in tumor cell lines.

A profound cytotoxic action of the antimalarial, artesunate (ART), was identified against 55 cancer cell lines of the U.S. National Cancer Institute (NCI). The 50% inhibition concentrations (IC50 values) for ART correlated significantly to the cell doubling times (P = 0.00132) and the portion of cells in the G0/G1 (P = 0.02244) or S cell cycle phases (P = 0.03567). We selected mRNA expression data of 465 genes obtained by microarray hybridization from the NCI data base. These genes belong to different biological categories (drug resistance genes, DNA damage response and repair genes, oncogenes and tumor suppressor genes, apoptosis-regulating genes, proliferation-associated genes, and cytokines and cytokine-associated genes). The constitutive expression of 54 of 465 (=12%) genes correlated significantly to the IC50 values for ART. Hierarchical cluster analysis of these 12 genes allowed the differentiation of clusters with ART-sensitive or ART-resistant cell lines (P = 0.00017). For exemplary validation, cell lines transduced with 3 of the 12 genes were used to prove a causative relationship. The cDNAs for a deletion-mutated epidermal growth factor receptor (EGFR) and for gamma-glutamylcysteine synthetase increased resistance to ART. The conditional expression of the CDC25A gene using a tetracycline repressor expression vector increased sensitivity toward ART. Multidrug-resistant cells differentially expressing the MDR1, MRP1, or BCRP genes were not cross-resistant to ART. ART acts via p53-dependent and- independent pathways in isogenic p53+/+ p21WAF1/CIP1+/+, p53-/- p21WAF1/CIP1+/+, and p53+/+ p21WAF1/CIP1-/- colon carcinoma cells.

Specific patterns of DNA copy number gains and losses in eight new glioblastoma multiforme cell lines.

Eight cell lines newly established from glioblastoma multiforme were examined by comparative genomic hybridization for their patterns of genomic imbalance. The total number of DNA copy number alterations (CNAs) found in the eight cell lines varied between 15 and 24. This characterized the examined cell lines (or the tumors they were derived from) as distinctly progressed in karyotypic evolution. The most frequent CNAs were gains of the entire chromosome 6 or, at least, parts of it, and of 7p22, which were found in all eight cell lines. Other changes present in seven of the eight cell lines were gains of 3q26qter and the entire chromosome 7 and losses of segments on chromosome 4q (e.g., 4q34q35) and of the short arm of chromosome 10. Enh(3q21q25), dim(4q22q33) and dim(4qter), dim(13q22), enh(15q14), and enh(18q22q23) were found in six of the eight cell lines. Several other CNAs [e.g., dim(9p21)] were found in common in five or less of the eight lines. Using a hierarchical cluster analysis, the specific patterns of genomic imbalance allowed various groupings of the examined cell lines. Although a close relation could be confirmed among all examined lines on the basis of shared CNAs, two main groups could be roughly differentiated. Among those there were also more or less closely related subgroups. However, also alterations which were restricted to one single cell line each were found, e.g., dim(1q41qter), dim(2q22qter), enh(4p), dim(5p), dim(4p13pter), dim(8q21qter), enh(9p), dim(9q), dim(11p14pter), enh(12q15q23), enh(13q21), dim(14q21qter), dim(15q21qter), dim(19q), and enh(22q). The comparison of the obtained data on gains and losses of DNA copy numbers in specific chromosomal segments with the data on localization of genes possibly associated with the biology of glioblastoma multiforme additionally shows high conformity but also disparity of the examined cell lines among each other, as well as compared to primary glioblastoma multiforme. Eventually, each of the cell lines could be characterized by its specific pattern of genomic imbalance.

Analysis of single nucleotide polymorphism C3435T of the multidrug resistance gene MDR1 in acute lymphoblastic leukemia.

Multidrug resistance is an important mechanism responsible for refractoriness of leukemia and worse outcome of patients. Overexpression of the multidrug resistance gene, MDR1, is of prognostic relevance in acute myeloid leukemia, while its role in acute lymphoblastic leukemia (ALL) is still under debate. Single nucleotide polymorphisms (SNP) have been detected in the MDR1 gene. The C3435T polymorphism in this gene seems to have functional and clinical consequences. In the present investigation, we have analyzed the role of the C3435T SNP for drug resistance and prognosis of human ALL. The C3435T SNP was analyzed in 20 T-ALL cell lines and in blood samples from 53 ALL patients and 7 healthy donors. The cell line panel consisted of cell lines not prior exposed in vitro to cytostatic drugs as well as of drug-resistant lines which were selected in vitro by exposure to doxorubicin, vincristine, methotrexate, or hydroxyurea. We have developed a highly sensitive matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based genotyping approach to survey the C3435T SNP. Furthermore, mRNA expression was determined by real time reverse-transcribed polymerase chain reaction and doxorubicin sensitivity by a growth inhibition assay. Surprisingly, we did not find a significant correlation between C3435T homo- or heterozygote genotypes and MDR1 mRNA expression of cell lines or blood samples from patients and healthy donors. Furthermore, there was no relationship between the response of the cell lines to doxorubicin and the C3435T genotypes. Homo- or heterozygosity did not correlate to survival of patients in the Kaplan-Meier analysis. In conclusion, we do not have reason to assume that the C3435T SNP contributes to drug resistance of ALL and prognosis of ALL patients.

Molecular modes of action of cephalotaxine and homoharringtonine from the coniferous tree Cephalotaxus hainanensis in human tumor cell lines.

Homoharringtonine (HHT) is an ester of cephalotaxine (CET), both of which derive from the Chinese coniferous tree Cephalotaxus hainanensis. HHT inhibited tumor cell growth at molar ranges comparable to established cytostatic drugs, whereas CET was 3-4 orders of magnitude less active. Inhibition concentration 50% (IC50) values of CET and HHT were significantly correlated to doxorubicin, vincristine, methotrexate, cisplatin, or camptothecin in 55 cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, Bethesda, Md., USA). We tested both drugs for resistance of cell lines which selectively overexpress the multidrug resistance (MDR)-conferring genes P-glycoprotein/ MDR1 (CEM/ADR5000), MDR-related protein 1 MRP1 (HL60/AR), and breast cancer resistance protein BCRP (MDA-MB-231-BCRP). A threefold and ninefold resistance to HHT and CET, respectively, was found in CEM/ADR5000 cells, while the other MDR cell lines did not show cross-resistance compared to their drug-sensitive counterparts. As the tumor suppressor p53 is another important factor of chemoresistance, we also analyzed the possibility that p53 affects the response of tumor cells to CET and HHT. Comparing the p53 mutational status of the 55 NCI cell lines (http://dtp.nci.nih.gov) with the IC50 values showed a significant correlation. Thus, CET and HHT were more active in cell lines without p53 mutation. We correlated the IC50 values of CET and HHT with the cell doubling times of the 55 NCI cell lines as proliferation parameter and observed that rapidly growing cells were more susceptible than slowly growing cell lines. We conducted a search mining the NCI's database for the mRNA expression of 465 genes in 55 cell lines and correlated the data with the IC50 values for CET and HHT. Of these genes 61 (=13%) correlated with the IC50 values for CET and 122 (=26%) with the IC50 values for HHT indicating the multifactorial mode of action of these drugs in cancer cells. We have chosen one example from these genes to test a causative role for drug response. U-87MG.DeltaEGFR cells transfected with an epidermal growth factor receptor ( EGFR) gene truncated in its extracellular domain through a deletion of exons 2-7 (Delta EGFR) were 14-fold more resistant to HHT than control cells transfected with mock expression vector or non-transfected cells. The present investigation presents a starting point to dissect the genes and molecular pathways involved in the tumor cells' response to CET and HHT in greater detail.

Genomic imbalances in drug-resistant T-cell acute lymphoblastic CEM leukemia cell lines.

Ten T-cell acute lymphoblastic (T-ALL) CEM cell lines selected for resistance toward methotrexate (CEM/MTX60PGA, CEM/MTX140LV, CEM/MTX1500LV, CEM/MTX5000PGA, CEM/MTXR1, CEM/MTXR2, and CEM/MTXR3), doxorubicin (CEM/ADR5000), vincristine (CEM/VCR1000), or hydroxyurea (CEM/HUR90), respectively, and parental drug-sensitive CCRF-CEM cells were analyzed using comparative genomic hybridization. Most genomic imbalances were not specific for drug resistance, as they were found in both parental and drug-resistant lines. Three aberrations were common to all or most cell lines analyzed: dim(5q35), dim(9p21p24), and enh(20q). We were concerned on those imbalances which were specifically present in drug-resistant but not in drug-sensitive cells. All methotrexate-resistant cell lines were characterized by an enhancement or an amplification of 5q13. The methotrexate resistance-conferring dihydrofolate reductase (DHFR) gene is located at this locus. Gain of DHFR was verified by PCR analyses. CEM/MTX60PGA, CEM/MTX140LV, CEM/MTX1500LV, and CEM/MTX5000PGA showed enh(14q21qter) and CEM/MTX5000PGA amp(5p13p15.2). These two loci harbor the methylenetetrahydrofolate dehydrogenase (MTHFD1) and 5'-methyltetrahdrofolate-homocysteine methyltransferase reductase (MTRR) genes, both of which are involved in folate metabolism. Their gain indicates a role in methotrexate resistance. A loss of 4q35 was found in CEM/MTXR2, CEM/MTXR3, and CEM/ADR5000 where the proapoptotic caspase-3 gene is located. The thioredoxin (TXN) locus 9q31 was enhanced in CEM/ADR5000 and CEM/MTX5000PGA cells. 2p22pter was increased in hydroxyurea-resistant CEM/HUR90 cells. Ribonucleotide reductase polypeptide M2 (RRM2), which confers resistance to hydroxyurea, resides at this locus. Other specific genomic imbalances in drug-resistant cell lines were dim(1p36.5), enh(4p), dim(8p22pter), enh(12p13), dim(17p), enh(18q12), enh(21q22.2), dim(21q22.2), and dim(22q13). All genomic imbalances were subjected to hierarchical cluster analysis and clustered image mapping to identify profiles of chromosomal aberrations in the cell lines. The obtained dendrograms allowed separation of imbalances common to all or most cell lines from other more individual aberrations. Furthermore, methotrexate-resistant cell lines clustered together. Our future efforts will be directed toward those imbalances which implicate still unknown candidate drug resistance genes.