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Martha S Bianchi

Publications and source records attributed to Martha S Bianchi.

12 recordsLinked to original sources

Telomeres, interstitial telomeric repeat sequences, and chromosomal aberrations.

Telomeres are specialized nucleoproteic complexes localized at the physical ends of linear eukaryotic chromosomes that maintain their stability and integrity. The DNA component of telomeres is characterized by being a G-rich double stranded DNA composed by short fragments tandemly repeated with different sequences depending on the species considered. At the chromosome level, telomeres or, more properly, telomeric repeats--the DNA component of telomeres--can be detected either by using the fluorescence in situ hybridization (FISH) technique with a DNA or a peptide nucleic acid (PNA) (pan)telomeric probe, i.e., which identifies simultaneously all of the telomeres in a metaphase cell, or by the primed in situ labeling (PRINS) reaction using an oligonucleotide primer complementary to the telomeric DNA repeated sequence. Using these techniques, incomplete chromosome elements, acentric fragments, amplification and translocation of telomeric repeat sequences, telomeric associations and telomeric fusions can be identified. In addition, chromosome orientation (CO)-FISH allows to discriminate between the different types of telomeric fusions, namely telomere-telomere and telomere-DNA double strand break fusions and to detect recombination events at the telomere, i.e., telomeric sister-chromatid exchanges (T-SCE). In this review, we summarize our current knowledge of chromosomal aberrations involving telomeres and interstitial telomeric repeat sequences and their induction by physical and chemical mutagens. Since all of the studies on the induction of these types of aberrations were conducted in mammalian cells, the review will be focused on the chromosomal aberrations involving the TTAGGG sequence, i.e., the telomeric repeat sequence that "caps" the chromosomes of all vertebrate species.

Animals↗

A comparative analysis of bleomycin-induced incomplete chromosome elements in two mammalian cell lines using a telomeric PNA probe.

Fluorescence in situ hybridization (FISH) with a telomeric peptide nucleic acid probe was employed to analyze the induction of incomplete chromosome elements (ICE; i.e., incomplete chromosomes and terminal fragments) by bleomycin (BLM) in two mammalian cell lines. Chinese hamster embryo cells (CHE cell line, average 2n = 23) and domestic rabbit cells (CPC cell line, average 2n = 44) were treated with 2.5 micro g/ml BLM; after 18 hr of incubation, first-division metaphases were stained with the telomeric probe, and ICE and other unstable chromosomal aberrations were scored. BLM induced ICE, dicentrics, and interstitial acentric fragments in CHE cells, but only ICE in CPC cells. About 50% of the metaphases in BLM-treated CHE cells contained one or more pairs of ICE, while only 20% of treated CPC cells contained ICE. Almost 100% of the BLM-induced ICE in both cell lines consisted of pairs formed by an incomplete chromosome and a terminal fragment. Our results confirm that ICE are the most frequent type of unstable chromosomal aberration induced by BLM in mammalian cells. Moreover, the present study shows that an increase in the chromosome number does not necessarily result in an increase in the frequency of BLM-induced ICE. The results also show that the difference in the chromosomal sensitivity to BLM in CHE and CPC cells is due to differences in the absolute frequency but not in the pattern (i.e., type and proportion) of ICE.

Animals↗

Analysis of streptozotocin-induced incomplete chromosome elements and excess acentric fragments in Chinese hamster cells using a telomeric PNA probe.

Fluorescence in situ hybridization (FISH) with a telomeric peptide nucleic acid (PNA) probe was employed to analyze the induction of incomplete chromosome elements (ICE, i.e., unjoined or "open" chromosome elements with telomeric signal at only one end) and excess acentric fragments (i.e., in excess of fragments resulting from the formation of dicentric and ring chromosomes) by the methylating agent streptozotocin (STZ) in a Chinese hamster embryo (CHE) cell line. CHE cells were treated with 0-4 mM STZ and chromosomal aberrations were analyzed in the first mitosis after treatment using the telomeric probe. Centric (incomplete chromosomes) and acentric (terminal fragments) ICE were the only unstable chromosome-type aberrations induced by STZ in CHE cells. The induction of these aberrations exhibited a curvilinear concentration-response relationship. About 40% of the metaphases present in cell cultures treated with STZ contained one or more pairs of ICE. In STZ-treated cells, ICE were always observed as pairs consisting of an incomplete chromosome and a terminal fragment. Moreover, all of the excess acentric fragments induced by STZ were of terminal type. These results indicate that chromosomal incompleteness is a very common event following exposure to STZ and suggest that all of the excess acentric fragments induced by STZ originate from terminal deletions.

Animals↗

Detection of incomplete chromosome elements and interstitial fragments induced by bleomycin in hamster cells using a telomeric PNA probe.

The detection of incomplete chromosome elements (ICE, i.e., elements with telomeric signal at only one terminal end) and interstitial fragments induced by the radiomimetic compound bleomycin (BLM) was carried out in a Chinese hamster embryo (CHE) cell line using FISH with a telomeric peptide nucleic acid (PNA) probe. CHE cells were treated with 0, 1, 2.5, 5, and 7.5 microg/ml of BLM and chromosomal aberrations were analyzed in the first mitosis after treatment using a telomeric PNA probe. The relationship between chromosomal aberrations frequency and bleomycin concentration was of linear type (P < 0.05 for all type of aberrations analyzed, i.e., multicentric chromosomes, centric rings, interstitial fragments and ICE). After BLM treatment, about 20-30% of the analyzed metaphases contained one or more pairs of ICE. Acentric interstitial fragments, lacking telomeric signals, were observed with a frequency of about 4-7 times higher than the dicentric frequency. Acentric interstitial fragments and ICE were induced at similar frequencies, except for the lowest BLM concentration (1 microg/ml), where the latter ones showed a higher frequency than the former ones. Furthermore, it was estimated that about 53% of excess acentric fragments originate from complete exchanges (interstitial deletions) and 47% from incomplete exchanges or terminal deletions. These results show that interstitial fragments and ICE are the most frequent asymmetrical chromosomal aberrations induced by BLM and indicate that true incompleteness is a common event following exposure to BLM. Moreover, the comparable trend of the concentration-response relationship for the different aberrations strongly suggests that all BLM-induced asymmetrical aberrations are formed by a similar underlying mechanism.

Animals↗

Analysis of streptonigrin-induced incomplete chromosome elements and interstitial fragments in Chinese hamster cells using a telomeric PNA probe.

We investigated the induction of incomplete chromosome elements (ICEs; i.e., elements with a telomeric signal at only one terminal end) and interstitial fragments induced by the antibiotic streptonigrin (SN) in a Chinese hamster embryo (CHE) cell line using FISH with a telomeric peptide nucleic acid probe. CHE cells were treated with 0-250 ng/ml SN and chromosomal aberrations were analyzed in the first mitosis after treatment using the telomeric probe. Exposure of CHE cells to SN resulted in a linear concentration-related increase in all of the aberration types analyzed (P < 0.05) except ring chromosomes. Depending on the SN concentration employed, 33-68% of the metaphases contained one or more pairs of ICEs (an incomplete chromosome accompanied by a terminal fragment or two incomplete chromosomes accompanied by a compound fragment). Pooled data from all SN concentrations revealed that 77.8% of the acentric fragments were terminal fragments, 18.8% interstitial fragments, and 3.4% compound fragments. Furthermore, it was estimated that about 80% of excess acentric fragments induced by SN originated from incomplete exchanges or terminal deletions and 20% from complete exchanges (interstitial deletions). These results show that incomplete chromosomes and terminal fragments are the most frequent asymmetrical chromosomal aberrations induced by SN and indicate that true incompleteness is a very common event following exposure to SN.

Animals↗

Effect of recombinant interferon-alpha on streptonigrin-induced chromosome aberrations and sister-chromatid exchanges in hamster cells.

The effect of recombinant interferon-alpha-2a (rIFN-alpha-2a) on the induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) by the radiomimetic antibiotic streptonigrin (SN, 250 ng/ml, 20 min, 37 degrees C) in Chinese hamster ovary (CHO) cells was investigated. Recombinant IFN-alpha-2a (4500-45,000 IU/ml) was added to the cell cultures 30 min before SN and left in the culture medium until the end of SN treatment or until cell harvesting. A statistically significant increase in the frequency of CAs and SCEs was observed following treatment with SN (P < 0.05), whereas treatments with rIFN-alpha-2a alone did not produce any significant increase of CAs and SCEs over control values. Low rIFN-alpha-2a doses produced a reduction in the frequency of CAs and an increase in the yield of SCEs induced by SN, while high doses of the cytokine caused an increase in the yield of CAs and a reduction in the frequency of SCEs induced by the antibiotic. In addition, rIFN-alpha-2a caused a marked inhibition (around 50%) on the yield of SN-induced chromatid-type aberrations in the G(2) phase of the cell cycle. It is suggested that the inhibitory effect of rIFN-alpha-2a on the SN-induced chromosome damage is due to the stimulation of DNA synthesis and repair by the cytokine. On the other hand, our results give further support to our previous hypothesis that the induction of CAs and SCEs by SN is based on different mechanisms.

Animals↗

Clastogenic effects of streptozotocin on human colon cancer cell lines with gene amplification.

We investigated the clastogenic effects of the methylating agent streptozotocin (STZ) on two human colon cancer cell lines. COLO320DM and COLO320HSR are cell lines derived from a human neuroendocrine colon carcinoma. STZ produced a dose-dependent increase in the frequency of chromosomal aberrations in both cell lines (p < 0.05) and induced fragmentation and/or pulverization of COLO320DM and COLO320HSR chromosomes. This effect was dose and time dependent. Severe chromosome damage was also found in cells that had progressed beyond the first metaphase, and a higher percentage of metaphases showing pulverized chromosomes were found in cells after the second than after the first division. This seems to indicate that STZ has a persistent and delayed clastogenic effect on COLO320DM and COLO320HSR cells. In addition, STZ produced a marked depression of the mitotic index in both cell lines. These results demonstrate that human colon cancer cell lines COLO320DM and COLO320HSR are highly sensitive to STZ, and suggest that this antibiotic has a good potential as a chemotherapeutic agent for colon cancer.

Antibiotics, Antineoplastic↗

Effect of recombinant interferon-alpha on bleomycin-induced chromosome damage in hamster cells.

The effect of recombinant interferon-alpha-2a (rIFN-alpha-2a) on the induction of chromosomal aberrations (CAs) by the radiomimetic antibiotic bleomycin (BLM, 5 microg/ml, 30 min, 37 degrees C) in Chinese hamster ovary (CHO) cells was investigated. Recombinant IFN-alpha-2a (4500-180,000IU/ml) was added to the cell cultures 0.5 or 24h before BLM (and left in the culture medium until the end of treatments) or immediately after BLM treatment (and left in the culture medium until harvesting). Cells were sampled at 18 or 2.5h after the end of treatments, in order to determine, respectively, the effect of rIFN-alpha-2a on the total chromosome damage induced by BLM and on the chromosome damage induced by this antibiotic in the G(2) phase of the cell cycle. A statistically significant increase in the frequency of CAs was observed following treatment with BLM (P<0.05), whereas treatments with rIFN-alpha-2a alone did not produce any significant increase of CAs over control values (P>0.05). The yield of CAs by BLM was significantly inhibited by rIFN-alpha-2a (P<0.05, 65.3% maximum inhibition). A strong inhibitory effect (around 80%) of rIFN-alpha-2a on the yield of BLM-induced CAs in the G(2) phase of the cell cycle was also observed. It is suggested that the inhibitory effect of rIFN-alpha-2a on the induction of CAs by BLM is mainly due to the stimulation of DNA synthesis and repair by the cytokine.

Animals↗

Chromosomal response of human lymphocytes to streptozotocin.

The induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) by the methylating agent streptozotocin (STZ) and the effect of this compound on mitotic index (MI) and cell cycle progression in human lymphocytes were investigated. Unstimulated (G(0)) or cycling lymphocytes derived from whole blood or purified lymphocyte cultures were pulse-treated with increasing doses of STZ for 0.5-24h. Induction of CAs by STZ was only observed in cycling lymphocytes derived from whole blood cultures (WBC) (P<0.05). On the contrary, STZ produced a significant and dose-response increase in the yield of SCEs in unstimulated as well as cycling lymphocytes (P<0.05). In addition, STZ induced a dose-dependent decrease in the MI but had a slight effect on cell cycle progression. These results suggest that SCEs are the most sensitive endpoint for evaluating the chromosomal effects of STZ on these cells.

Cell Cycle↗

Mosaic AZF deletions and susceptibility to testicular tumors.

We tested for azoospermia factor (AZF) deletions 17 loci corresponding to AZF subintervals a-d in 17 cases of testicular tumors occurring in Finns. While DNA samples from 48 CEPH and 32 Finnish males showed no deletions, patients with testicular cancer displayed AZF deletion mosaicisms in various non-tumor tissues (13 cases) and specific deletion haplotypes in tumor tissues (10 cases). Two of the cases with AZF deletions were testicular non-Hodgkin lymphomas indicating that Y-microdeletions appear also in malignancies other than seminoma and non-seminoma tumors. In good agreement with this assumption, we detected one AZF deletion in normal cells from 1 of 5 HNPCC cases, heterozygous for an MLH1 mutation. We propose that AZF deletions occur in early embryogenesis due to mutations of TSPY, mismatch repair (MMR), or X-specific genes. Since fathers of testicular, tumor cases did not exhibit AZF deletions, we assumed they were not carriers of the mutation inducing AZF deletion-mosaicisms. Therefore, tumor cases should have received the MMR gene or X mutations via the maternal lineage, or for the case of TSPY and MMR genes via a sperm carrying a mutation occurred in the paternal germ-cell line. We consider AZF microdeletions in non-tumor cells to be part of a broader pattern of chromosome instability producing susceptibility to testicular tumors. Clonal transformation and expansion of one of these tumor-susceptible cell lineages give rise to testicular tumors showing genome anomalies characteristic of testicular cancers (i12p, LOH and genetic imbalance for various autosomal regions, Y- and autosomal MSI, specific AZF deletion haplotypes).

Base Pair Mismatch↗

Genotoxicity of streptozotocin.

Streptozotocin (Streptozocin, STZ, CAS No. 18883-66-4) is a monofunctional nitrosourea derivative isolated from Streptomyces achromogenes. It has broad spectrum antibiotic activity and antineoplastic properties and is often used to induce diabetes mellitus in experimental animals through its toxic effects on pancreatic beta cells. STZ is a potent alkylating agent known to directly methylate DNA and is highly genotoxic, producing DNA strand breaks, alkali-labile sites, unscheduled DNA synthesis, DNA adducts, chromosomal aberrations, micronuclei, sister chromatid exchanges, and cell death. This antibiotic was found to be mutagenic in bacterial assays and eukaryotic cells. STZ is also carcinogenic; a single administration induces tumors in rat kidney, liver, and pancreas. Several lines of evidence indicate that free radicals are involved in the production of DNA and chromosome damage by this compound. Because of the use of STZ as an antineoplastic agent, the study of its genotoxicity has considerable practical significance. The purpose of this review is to present our current knowledge regarding the genotoxicity of STZ.

Animals↗