Thomas Efferth

Homozygous deletions of CDKN2A caused by alternative mechanisms in various human cancer cell lines

The CDKN2A tumor‐suppressor locus on chromosome band 9p21, which encodes p16INK4A, a negative regulator of cyclin‐dependent kinases, and p14ARF1, an activator of TP53, is inactivated in many human cancers by point mutation, promoter hypermethylation, and, often, deletion. Homozygous deletions are unusually prevalent at this locus in very different human cancers. In the present study, we compared deletions in squamous cell carcinoma of the head and neck (SCCHN) cell lines to those in T‐cell acute lymphatic leukemia (T‐ALL), glioma, and bladder carcinoma (TCC) cell lines. Of 14 SCCHN lines, 10 showed homozygous deletions of CDKN2A, one displayed promoter hypermethylation with gene silencing, and one had a frameshift deletion in exon 2. Many deletion ends were in or proximal to the repetitive sequence clusters flanking the locus. Breakpoint junctions displayed variable microhomologies or insertions characteristic of DNA repair by nonhomologous end‐joining. In general, deletions were much smaller in SCCHN than in TCC and glioma. In T‐ALL, breakpoints were near consensus sites for recombination mediated by RAG (recombination activating genes) enzymes, and the structure of the junctions was consistent with this mechanism. We suggest that different mechanisms of CDKN2A deletion prevail in different human cancers. Aberrant RAG‐mediated recombination may be responsible in T‐ALL, and exuberant DNA repair by nonhomologous end‐joining is the likely prevailing mechanism in SCCHN, but a distinct mechanism in TCC and glioma remains to be elucidated.

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 Kendalls 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.

Development of Drug Resistance in Trichomonas vaginalis and its Overcoming with Natural Products

Trichomoniasis is an infectious disease afflicting women worldwide. The protozoan parasite Trichomonas vaginalis is the causative agent of this sexually-transmitted disease, including also men in its infection cycle. The disease is usually not life-threatening, but has been associated with the development of cervical cancer and increased susceptibil- ity to HIV. Approved drugs are 5-nitroimidazoles, with metronidazole being the drug of first choice. These drugs act via induction of oxidative stress and DNA-damage, leading to cell death in the parasite. Nevertheless, with the development of resistant T. vaginalis strains the treatment of the disease becomes exceedingly difficult. Mechanisms of drug resistance are characterized by reduced expression or even loss of proteins necessary for drug activation and a decreased reductive nature in the parasite. A promising strategy for research into new drugs and moreover, to overcome drug resistance, are compounds derived from natural sources. The present study provides a summary of all so far investigated small molecules with antitrichomonal activity; promisingly, some also show efficacy against resistant strains. Whereas the list of chemically characterized compounds derived from plants is rather short, literature provides immense applications of crude plant extracts tested against T. vaginalis. This demonstrates the absence of studies in this field aimed to identify and isolate single natural products exhibiting antitrichomonal features. Likewise, elucidating their mode of action on a molecular basis is of paramount importance.

Comparative M-FISH and CGH analyses in sensitive and drug-resistant human T-cell acute leukemia cell lines.

Cell lines of human T-cell acute lymphoblastic leukemias (T-ALL) have gained high interest for study of mechanisms of cytostatic drug resistance. However, they should also be suited to examine the validity and reliability of molecular cytogenetic techniques in detecting genomic alterations in neoplastic cells. Therefore, comparative genomic hybridization (CGH) and 24-color-fluorescence-in-situ-hybridization (M-FISH) were applied to eight sublines of CCRF-CEM leukemia cells selected in vitro for drug resistance and to their drug-sensitive parental counterparts. All cell lines were characterized by altered chromosome numbers and by a variety of chromosomal structural aberrations as shown by M-FISH. The great majority of anomalies detected by this technique were confirmed by CGH. Interestingly, a considerable number of the rearrangements found were imbalanced. Amplifications of 5q13 in the six methotrexate-resistant cell lines, a del(9)(p21pter) in all lines examined, and a gain of chromosome 20 in 9 of the 10 lines examined were readily detected by both techniques. The same held true for losses of chromosomes 17 and 18 in the near tetraploid cell lines which could also be confirmed by CGH. Some imbalances of genomic material detected by CGH were, however, not observed by means of M-FISH, possibly due to the limited extension of the corresponding chromosomal segment involved or the small subpopulation of cells affected. On the other hand, reciprocal translocations, balanced isochromosomes, and small deletions remained mainly undetected by CGH. A comparison of chromosomal alterations in drug-resistant and parental cell lines showed not only amplifications of chromosomal segments harboring well-known drug resistance genes, e.g., the dihydrofolate reductase gene, but also chromosomal changes which may involve novel genes associated with drug resistance. Thus, the present study has clearly unveiled the strengths and weaknesses of both techniques which can excellently complement each other. Their combination allowed a distinct improvement of the definition of the complex karyotypes of drug-resistant cell lines.

Objectifying Acupuncture Effects by Lung Function and Numeric Rating Scale in Patients Undergoing Heart Surgery

Rationale. Poststernotomy pain and impaired breathing are common clinical problems in early postoperative care following heart surgery. Insufficiently treated pain increases the risk of pulmonary complications. High-dose opioids are used for pain management, but they may cause side effects such as respiratory depression. Study Design. We performed a prospective, randomized, controlled, observer-blinded, three-armed clinical trial with 100 patients. Group 1 (n = 33) and Group 2 (n = 34) received one 20 min session of standardized acupuncture treatment with two different sets of acupoints. Group 3 (n = 33) served as standard analgesia control without additional intervention. Results. Primary endpoint analysis revealed a statistically significant analgesic effect for both acupuncture treatments. Group 1 showed a mean percentile pain reduction (PPR) of 18% (SD 19, P < 0.001). Group 2 yielded a mean PPR of 71% (SD 13, P < 0.001). In Group 1, acupuncture resulted in a mean forced vital capacity (FVC) increase of 30 cm3 (SD 73) without statistical significance (P = 0.303). In Group 2, posttreatment FVC showed a significant increase of 306 cm3 (SD 215, P < 0.001). Conclusion. Acupuncture revealed specific analgesic effects after sternotomy. Objective measurement of poststernotomy pain via lung function test was possible.