Walter Bardenheuer

Analysis of the p53 gene in patients with isochromosome 17q and Ph1-positive or -negative myeloid leukemia

Increased incidence of p53 gene aberrations or chromosome 17p monosomy resulting from an isochromosome 17q [i(17q)] has been observed with transition of chronic myelogenous leukemia (CML) to myeloid blast crisis (BC), and in some patients with poor risk acute myeloid leukemia (AML) progressing from myelodysplastic syndrome (MDS). These data suggested that disease progression may be linked to bi-allelic inactivation of p53. Here, we report on p53 gene analyses of nine patients with CML-BC and AML who showed an i(17q) as characteristic cytogenetic anomaly. Using Southern blots, agarose gel electrophoresis and single-strand conformation polymorphism analyses of PCR products from genomic DNA and cDNA, spanning exons 4 through 9, we did not detect any structural abnormalities of the remaining p53 allele. These findings question the hypothesis that p53 gene alterations are the principal molecular event responsible for progression of CML chronic phase or MDS to i(17q)-positive CML-BC or AML, respectively.

The p44(S10) locus, encoding a subunit of the proteasome regulatory particle, is amplified during progression of cutaneous malignant melanoma

Gene amplification is frequently present in human tumors, although specific target genes relevant to many amplified loci remain unidentified. An expression cloning assay enabled identification of a candidate oncogene derived from human chromosome 3p14.1. The cDNA retrieved from morphologically transformed cells contained the full-length protein coding region and detected an abundant transcript in the same cells. Sequence analysis revealed identity with the wild-type sequence of p44S10, a highly conserved subunit of the 26S proteasome that exhibits similarity to the Arabidopsis fus6/cop11 family of signaling molecules. p44S10 gene copy number and mRNA expression were increased in association with segmental 1.8–11-fold chromosomal gains in cutaneous malignant melanoma cell lines (5/13; 40%) and tumors (2/40; 5%), and in breast cancer MCF-7 cells. Likewise, malignant progression of human radial growth phase WM35 melanoma cells was associated with amplification and increased expression of endogenous p44S10, and increased expression of p44S10 was sufficient to induce proliferation of WM35 cells in vivo. The results demonstrate segmental copy number gains within chromosome 3p in cutaneous malignant melanoma and suggest that deregulation of a proteasome regulatory particle subunit may contribute to the malignant phenotype.

Minimal deletion of 3p13-->14.2 associated with immortalization of human uroepithelial cells.

Immortalization and tumorigenic transformation of many human cell types, including human uroepithelial cells (HUCs), are frequently associated with loss of genetic material from the short arm of chromosome 3 (3p). In addition, losses of 3p have been observed in many human cancers including renal cell carcinoma, lung cancer, breast cancer, and bladder cancer. Genetic studies suggest that there are at least two regions on 3p in which tumor suppressor genes might be located, but the precise location of these genes is not known. We studied chromosome 3 losses that were specifically associated with immortalization of five independent human papilloma virus 16 (HPV16) E6‐ or E7‐transformed HUCs. Cytogenetic analysis showed that the smallest common region of deletion was 3p14.1→14.2. Fluorescence in situ hybridization using a 3p13→14‐specific yeast artificial chromosome (YAC) contig showed the precise localization of the breakpoints to be in 3p13 and 3p14.2, thus defining the smallest common overlap of 3p deletions in HPV16 E6‐ or E7‐immortalized HUCs. These results suggest the presence in this region of genes involved in the control of senescence in vitro and possibly tumorigenesis in vivo. Genes Chromosomes Cancer 21:39–48, 1998.

Protection of hematopoietic cells from O6‐alkylation damage by O6‐methylguanine DNA methyltransferase gene transfer: studies with different O6‐alkylating agents and retroviral backbones

Abstract: Overexpression of O6‐methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O6‐alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O6‐alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT‐cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus‐based retroviral vector (N2/Zip‐PGK‐MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony‐forming units (CFU) was demonstrated for all four drugs. In comparison to mock‐transduced controls, after transduction with N2/Zip‐PGK‐MGMT the IC50 for CFU increased on average 4.7‐fold for ACNU, 2.5‐fold for BCNU, 6.3‐fold for CCNU and 1.5‐fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT‐cDNA from a murine embryonic sarcoma virus LTR (MSCV‐MGMT) or a hybrid spleen focus‐forming/murine embryonic sarcoma virus LTR (SF1‐MGMT) were compared with the N2/Zip‐PGK‐MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1‐MGMT construct was most efficient especially at high ACNU concentrations (8–12 µg/ml). Similar results were obtained for protection of murine high‐proliferative‐potential colony‐forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer.

Identification of the full-length huntingtin- interacting protein p231HBP/HYPB as a DNA-binding factor.

Neurodegeneration in Huntingtons disease (HD) is associated with an elongated glutamine tract in the widely expressed huntingtin protein. Although the pathogenic mechanisms are still unknown, the distinct physical properties of mutant huntingtin in the brain suggest that other factors including huntingtin-interacting proteins might play a specific role. We have previously identified a DNA-binding motif in the proximal E1A promoter of adenovirus serotype 12 as responsible for E1A autoregulation. Here, we identified the p231HBP protein as a DNA-binding factor, the C-terminal portion of which has recently been characterized as the huntingtin-interacting protein HYPB of unknown function. We have determined the full-length cDNA sequence, identified several domains supporting its gene regulatory functions, and mapped the HBP231 gene to chromosome 3p21.2-p21.3. Our results provide an interesting molecular link between huntingtin and a DNA-binding factor, implicating that this interaction might result in the alteration of cellular gene expression involved in HD pathogenesis.

Hematoprotection by transfer of drug-resistance genes.

Myelosuppression represents a major side effect of cytotoxic anti-cancer agents. Infection due to granulocytopenia and the risk of bleeding due to thrombocytopenia compromise the potential of curative and palliative chemotherapy. Considering the many chemotherapeutic agents for which drug resistance genes have been described, and the recent improvements in vector and transduction technology, it seems conceivable that drug resistance gene transfer into a patient’s autologous hematopoietic stem or progenitor cells will be able to reduce or abolish chemotherapy-induced myelosuppression.

Identification of novel ‘expressed sequence tags’ within the FHIT gene locus in human chromosome region 3p14.2

Abstract Losses of genetic material within human chromosome regions (HCR) 3p12–p14 and 3p21–p22 are observed in various neoplasias, suggesting tumor suppressor gene (TSG) loci within these regions. HCR 3p14 is particularly interesting as it contains the t(3;8) translocation breakpoint of a hereditary renal cell carcinoma, the FRA3B fragile site, and DNA markers deleted in several types of human cancer. We here report on the identification of five novel ‘expressed sequence tags’ (ESTs) within 3p14.2 which map proximal to exon 9 of the candidate TSG, FHIT. These ESTs may be valuable for elucidation of the supposed TSG content in 3p14.2.