Annalisa Zunino

Study on aneuploidy and p53 mutations in astrocytonias

To determine whether a correlation exists between aneuploidy and p53 status in astrocytic tumors we analyzed 48 astrocytomas with different grades of malignancy for the presence of p53 mutations and aneuploidy of chromosomes 10 and 17 (Ch10, Ch17), known to be particularly involved with this type of tumor. We used polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis on exons 5-8 of the p53 gene, and fluorescence in situ hybridization (FISH) analysis on interphase nuclei using chromosome specific pericentromeric probes, respectively. Our results showed that Ch10/Ch17 aneuploidy is a common early event in astrocytomas (90% of low grade tumors are aneuploid). p53 mutations and Ch17 aneuploidy are early events, but their incidence is not dependent on tumor grade. Loss of Ch10 is the only alteration that significantly correlates with tumor progression. No significant correlation between the presence of Ch10/Ch17 aneuploidy and p53 mutations was found. However, the coexistence of p53 mutations and aneuploidy, was observed in a subset of cases. The presence of p53 mutations appeared to be a significant predictor of a poor prognosis. In conclusion, genomic instability may or may not be associated with p53 mutations in astrocytomas, thus suggesting that other cellular determinants can also be responsible for the aneuploidy observed.

Alkaline DNA fragmentation, DNA disentanglement evaluated viscosimetrically and sister chromatid exchanges, after treatment in vivo with nitrofurantoin

Nitrofurantoin was not positive as a carcinogen in long term assays. In vitro it was positive in some short term tests and negative in others. We have examined Nitrofurantoin for its capability of inducing DNA damage in vivo. With the alkaline elution technique, Nitrofurantoin appeared clearly positive in all the tissues examined (liver, kidney, lung, spleen and bone marrow). In the liver we also observed some cross-linking effect. In bone marrow cells Nitrofurantoin was also clearly positive in terms of sister chromatid exchanges (SCEs) induction. DNA damage in vivo was also examined with a viscosimetric method, more sensitive than alkaline elution. With this method the results were essentially negative, suggesting that the two methods detect different types of damage. In view of its positivity in many organs and in two short term tests in vivo, the carcinogenic potential of Nitrofurantoin should be reconsidered.

Exposure of human lymphocytes and lymphoblastoid cells to simulated microgravity strongly affects energy metabolism and DNA repair

Exposure of freshly drawn lymphocytes and lymphoblastoid cells (LB and COR3) to simulated microgravity decreased the intracellular ATP concentration to 50%–40% of the value found in normal growth conditions. The decrease was reversible although recovery to normal values occurred only slowly both in lymphocytes and in lymphoblastoid cells. Poly(ADP‐ribose) polymerase (PARP ) activity was increased indicating that cells exposed to conditions of reduced gravitation experience stress. Exposure to microgravity forces cells into a condition of metabolic quiescence in which they appear to be particularly sensitive to subsequent exposures to a genotoxic agent. Thus, treatment of cells with the strong redox agent potassium bromate under microgravity conditions, indicated an impairment in repair of DNA 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), an oxidized derivative of deoxyguanosine. We conclude that gravitational modulation of the kind routinely obtained under laboratory conditions and during spaceflights is a stressful process to which cells appear to be extremely sensitive. These effects may reflect the physiological alterations observed in astronauts and in animals following spaceflights or exposure to conditions of simulated microgravity.

Assay of phenacetin genotoxicity using in vitro and in vivo test systems

Phenacetin was assayed in a battery of five short-term tests. (1) In a DNA-repair test using various Escherichia coli strains, the drug was not directly genotoxic nor did it induce nonreparable DNA damage in the presence of rat liver S9 fractions, while it was weakly active following activation with hamster liver S9. (2) In the Ames reversion test (strains TA97, TA98, TA100, and TA102 of Salmonella typhimurium, phenacetin reverted only TA100, and only in the presence of hamster liver S9. Mutagenicity was related to the concentration both of the drug and of the above metabolic system. There was no activation with hamster kidney S9, uninduced chicken liver S9, or with a variety of liver S9 preparations from rats treated with enzyme inducers (Aroclor 1254, phenobarbital, or 3-methylcholanthrene) and/or glutathione depletors (diethyl maleate or buthionine sulfoximine). Hamster liver S9 compared favorably to rat and even more to chicken liver S9 fractions also in activating various promutagens [3-amino-1-methyl-SH-pyrido (4,3-b)-indole, 2-aminofluorene, aflatoxin B1, benzo[a]pyrene, and benzo[a]pyrene-trans-7,8-diol] and in decreasing the mutagenicity of direct-acting compounds (4-nitroquinoline N-oxide and sodium dichromate). (3) Phenacetin was borderline positive in a forward mutation test (6-thioguanine resistance) in V79 cells, only in the presence of hamster liver S9, and gave negative results in the presence of rat liver S9 or without any metabolic system. (4) Following in vivo treatment, the alkaline elution assay did not reveal any DNA fragmentation in bone-marrow cells of ip-treated mice or in liver cells of rats treated by gavage. Apparent DNA damage was instead observed in the kidneys of rats receiving the drug by gavage or in the liver following ip administration. However, the effect was prevented (liver) or reduced (kidney) by preliminary perfusion of the organs, which discards (liver) or makes uncertain (kidney) the hypothesis of a true in vivo DNA damage. (5) Phenacetin ip induced in mouse bone-marrow cells a poor yet statistically significant increase in sister chromatid exchanges.

Quantitative correlation between carcinogenicity and sister chromatid exchange induction in vivo for a group of 11 n‐nitroso derivatives

The quantitative correlation between induction of sister chromatid exchanges (SCEs) in vivo and carcinogenic potency was examined for 11 nitroso derivatives and was compared with the correlation of alkaline DNA fragmentation in liver DNA in vivo and with the Ames test. The correlation between DNA adducts and SCEs was also evaluated. DNA damage was slightly more predictive and the Ames test less predictive than SCE evaluation. The predictivity of these tests for this class of compounds was compared with the predictivity shown for different classes of chemical compounds.

Uncommon cytogenetic findings in a case of splenic marginal zone lymphoma with aggressive clinical course

The majority of splenic marginal zone lymphoma (SMZL) patients experience an indolent clinical course; however, some cases transform to a high-grade lymphoma. Cytogenetic analyses have shown that chromosome 7 is the most frequently altered chromosome and, in some cases, 7q deletion has been found as a single aberration, suggesting its association with the development of SMZL. We studied one patient showing clinical features of SMZL with an aggressive course. Immunophenotypic, conventional and molecular cytogenetic techniques were applied to support the diagnosis. The immunophenotype of peripheral blood mononuclear cells showed the presence of 90% B-lymphocytes. Cytogenetic analysis indicated the presence of a stem-line lacking normal chromosomes 7, but showing a der(7) and a ring, and a side-line with additional aberrations: t(2;22), add(8). Fluorescence in situ hybridization analysis revealed a loss of the 7q32 region. Nonclonal rearrangements involving chromosome 7 were also detected. Chromosome 7 rearrangements were studied to investigate their evolution during the development of the pathology. We have shown that in this patient both chromosomes 7 underwent different rearrangements leading to a loss of the 7q32 region and that the ring chromosome originated from chromosome 7 and was associated with a t(7;7) (p22;q31). We conclude that not only the 7q deletion but also the proneness of chromosome 7 to rearrange might have played a role in the progression of this SMZL.

Cytogenetic characterization and cell cycle analysis of three human colon adenocarcinoma cell lines: comparison between two- and three-dimensional cell culture systems.

ABSTRACT The aim of the study was to investigate whether changes in the pattern of gene copy number and cell cycle were present passing from the two- to the three-dimensional cell culture system. We used three human colon adenocarcinoma cell lines grown two- and three-dimensionally. We analyzed morphology, karyotype, chromosomal gain and losses, and cell cycle. In three-dimensional cell cultures the growth is delayed and arrested in G1 phase without specific rearrangements in the three-dimensional cultures compared to the two-dimensional cultures. These data suggest that the differences between the two- and three-dimensional cell culture systems do not involve chromosomal rearrangements.

Stable Formation of Mutated p53 Multimers in a Chinese Hamster Cell Line Causes Defective p53 Nuclear Localization and Abrogates Its Residual Function

We have previously described a methotrexate‐resistant cell line (MTX M) characterized by amplified dihydrofolate reductase (DHFR) genes, cytoplasmic p53 localization, and p53 stable tetramers. To investigate the p53 functionality in MTX M, the effect of chemical/physical agents was studied. In MTX M cells, DNA damage did not induce p53 or mdm‐2 protein, while in the parental V79 cells, a residual p53 activity was found. cDNA sequencing showed that V79 and MTX M cells share the same mutations, indicating that the complete loss of p53 function in MTX M cells was due to cytoplasmic sequestration of a mutated p53 with residual activity. In Chinese hamster, both p53 and DHFR genes map on short arm of chromosome 2 suggesting that p53 itself might be amplified. However, fluorescence in situ hybridization with a hamster p53 probe showed only a single signal. Thus, the presence of p53 stable tetramers in MTX M cells, although correlated with DNA amplification, could not be the consequence of either p53 or DHFR gene amplification. Expression of a C‐terminal human p53 peptide does not induce p53 nuclear accumulation, indicating that the cytoplasmic localization is due to a mechanism different from that already described in cancer cell lines. Treatments with Sodium Butyrate induced β‐tubulin polymerization, but did not apparently organize a normal microtubule network, which is shown to be important for the p53 localization. Our data indicated that in MTX M cells, p53 is sequestered in the cytoplasm by a novel mechanism that abrogates p53 residual function. J. Cell. Biochem.

Abstract 496: Isolation and characterization of a highly invasive subpopulation from MDA-MB-231 breast cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The acquisition of an invasive phenotype is a pre-requisite for metastasis. We set out to develop cellular systems that can mirror transient and stbale molecular alterations that confer an invasive phenotype to breast cancer cells. We observed that it is possible to isolate invasive subpopulations from moderately invasive cancer cell lines. Enrichment of invasive sub-populations of MDA-MB-231 breast cancer cells in three successive preparative invasion assays in Matrigel covered Boyden chambers yielded a highly invasive cell line. Prolonged cultivation of these cells did not abolish the invasive phenotype although not all phenotypic changes acquired during selection are maintained by long term cultures. Genetic analyses of these cells by cytogenetics and array based comparative genome hybridization revealed many genetic alterations including increased ploidy. The flow cytometric DNA Index (DI) changes from 1.28 to 2.28. Cells with DI 2.28 constitute 1.4% of the parental cell line. Whole genome SNP analysis shows that the two populations are genetically related excluding any cell contamination. The invasive cells proliferate and undergo apoptosis similar to the parental cells. Commitment to apoptosis is increased since invasive cells respond more strongly to curcumin or peroxide induced apoptosis. Invasive cells show relative resistance to the cytotoxic, alkylating agent Doxorubicin and increased sensitivity to the anti-mitotic drugs Vincristine and Taxol. Increased resistance to the topoisomerase II inhibitor Mitoxantrone is observed only transiently in invasive cells and lost in long term cultures. Similarly, the chemokines CXCL1 and -2 are transiently upregulated. Response to the anti-diabetic drug Metformin showed a reduced sensibility for invasive cells in terms of growth inhibition (IC50). Gene expression profiling shows complex alterations in gene expression. Many of the genes that are differentially expressed in highly versus moderately invasive cells are differentially expressed in human breast cancer cases with and without distant metastasis and correlate with disease free survival. The invasive phenotype is not related to stem cell features nor to epithelial mesenchymal transition. These cells constitute a novel model for tumor progression. Tumorigenicity and metastatic potential in vivo are currently being tested. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 496. doi:1538-7445.AM2012-496

Chromosomal assignment of human O6-methylguanine-DNA-methyltransferase gene by hamster–human somatic cell hybrids

Using an in vitro assay to measure O6-methylguanine-DNA-methyltransferase (MT) activity in cell extracts from a panel of human-hamster cell hybrids, we were able to locate the human MT gene on chromosome 10. Chinese hamster cells have little or no MT activity and the presence of human chromosome 10 was a necessary condition for MT activity in cell hybrids. In some cell hybrids carrying chromosome 10, however, MT activity was not higher than that of hamster cells. As an explanation for this result, genetic determinants repressing MT expression and/or activity might be present in other human chromosomes carried by MT-negative cell hybrids. Partial hyperploidy of the hamster karyotype, variable activity of the parental human cell lines and changes during subculturing of the cell hybrids might also account for the lack of enzymatic activity in chromosome 10 containing hybrids.