DNA and chromosome damage induced by bleomycin in mammalian cells: An update.

Bleomycin (BLM) is an antibiotic isolated from Streptomyces verticillus. It has radiomimetic actions on DNA thus it has been widely used in clinical chemotherapy for the treatment of different types of cancer, including head and neck tumors, lymphomas, squamous-cell carcinomas and germ-cell tumors. Because of this, the study of BLM genotoxicity is of practical interest. This antibiotic is an S-independent clastogen and an agent that generates free radicals and induces single- and double-strand breaks in DNA. In the present review, we will summarize our current knowledge concerning the DNA and chromosome damage induced by BLM in mammalian cells, with emphasis on new developments published since 1991.

Effect of thiol compounds on bleomycin-induced DNA and chromosome damage in human cells.

Non-protein thiols are considered radioprotectors, preventing DNA damage by ionizing radiation. As bleomycin (BLM) is a radiomimetic agent it was proposed that thiols may prevent DNA damage produced by this antibiotic. However, results obtained with thiols and BLM-combined treatments in living cells are contradictory. The goal of this work was to assess the influence of five non-protein thiols of different electrical charge and chemical composition, on the DNA damage, DNA repair, chromosomal aberrations and cell killing induced by BLM. We found that, at the chromosomal level and cell killing, Glutathione, ß-Mercaptoethanol and cysteine showed a protective effect, while ditiothreitol and cysteamine increased them, whereas at the DNA level all thiols potentiated the DNA damage induced by BLM, most probably due to a reactivation of the BLM complex.

Telomere instability is present in the progeny of mammalian cells exposed to bleomycin.

We analyzed the chromosomal aberrations involving telomeres in the progeny of mammalian cells exposed to the radiomimetic compound bleomycin (BLM) in order to determine if this antineoplastic drug induces long-term telomere instability. To this end, rat cells (ADIPO-P2 cell line, derived from adipose cells from Sprague-Dawley rat) were treated with a single concentration of BLM (2.5 µg/ml), and chromosomal aberrations were analyzed 18 h and 10 days after treatment by using PNA-FISH with a pan-telomeric probe [(TTAGGG)n repeats]. Cytogenetic analysis revealed a higher frequency of aberrations at 18 h and 10 days after treatment in BLM-exposed cultures vs. untreated cultures, although the yield of BLM-induced aberrations 10 days after treatment decreased about 25% compared with the one at 18 h after treatment. Moreover, the level of telomerase activity in BLM-treated cells compared with that of untreated control cells was significantly higher at 10 days after treatment, but did not differ at 18 h after treatment. These data indicate that in terms of unstable aberrations, the in vitro clastogenic effect of BLM on ADIPO-P2 cells persists for at least 10 days after exposure. In addition, our data demonstrate, for the first time, that BLM-induced telomere instability in mammalian cells (cytogenetically detectable as incomplete chromosome elements and telomere FISH signal loss and duplication) persists for several generations after exposure. Moreover, the appearance of telomere fusions in BLM-exposed cells 10 days after treatment suggests that this compound can induce delayed telomere instability. The increase in telomerase activity in BLM-exposed cells 10 days after treatment is accompanied by the presence of aberrations directly related to telomere dysfunction. This fact suggests that telomerase is not directly involved in BLM-induced telomere instability.

Streptonigrin induces delayed chromosomal instability involving interstitial telomeric sequences in Chinese hamster ovary cells.

We analyzed the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells exposed to the radiomimetic compound streptonigrin (SN), in order to determine whether interstitial telomeric sequences (ITSs) are involved in the long-term clastogenic effect of this antibiotic. CHO cells were treated with a single concentration of SN (100ng/ml), and the frequency of unstable chromosomal aberrations was determined at three times after treatment (18h, and 6 and 15 days) by using PNA-FISH with a pan-telomeric probe. Cytogenetic analysis revealed a higher frequency of aberrations at 18h and 6 days after treatment in SN-exposed cultures vs. untreated cultures. The percentage of damaged cells and the yield of SN-induced aberrations at 6 days after treatment increased on average twofold compared with the ones at 18h after treatment. Moreover, a significant decrease in the frequency of aberrations was observed in SN-exposed cells at 15 days after treatment, resulting in a frequency of aberrations significantly lower than the frequency of aberrations observed in the corresponding control cultures. These data indicate that SN induces delayed chromosomal instability in CHO cells, and that the in vitro clastogenic effect of this compound persists for at least 6 days but less than 15 days after treatment. In addition, we found that SN induces delayed ITSs instability, cytogenetically detectable as additional FISH signals and centromeric breaks involving dissociation of the telomeric signal 6 days after treatment. We propose that the delayed effect of SN on ITSs results from breakage of heterochromatic centromeric ITSs blocks and further insertion of these sequences at the sites of mono- or isochromatid breaks occurring at G2 or G1-S phases of the cell cycle, respectively, since most of the additional FISH signals were present as single or double dots, and located at interstitial sites of the involved chromosomes.

Bleomycin induces delayed instability of interstitial telomeric sequences in Chinese hamster ovary cells.

We analyzed the behavior of interstitial telomeric sequences (ITSs) in the progeny of Chinese Hamster Ovary (CHO) cells exposed to the radiomimetic compound bleomycin (BLM) in order to determine if ITSs play some role in the long-term clastogenic effect of this antibiotic. To this end, CHO cells were treated with a single concentration of BLM (2.5µg/ml), and the frequency of unstable chromosomal aberrations was determined at several times after treatment (18h, and 6, 15 and 34/36 days) by using PNA-FISH with a pan-telomeric probe [(TTAGGG)n repeats]. Cytogenetic analysis revealed a higher frequency of aberrations at 18h and 6 days after treatment in BLM-exposed cultures vs. untreated cultures, although the yield of BLM-induced aberrations decreased on average five times 6 days after treatment compared with the one induced 18h after treatment. Moreover, no significant differences in the frequency of aberrations were observed between untreated and BLM-exposed cells at 15 or 34/36 days after treatment. These data indicate that, in terms of unstable aberrations, the in vitro clastogenic effect of BLM on CHO cells persists for at least 6 days but less than 15 days after exposure. In addition, we found that BLM induces ITSs instability, cytogenetically detectable as acentric fragments (18h after treatment) or additional (new) FISH signals (6 days after treatment). We propose that the delayed effect of BLM on ITSs mainly results from breakage of heterochromatic ITSs blocks and further insertion of these sequences at the sites of monochromatid breaks occurring at G2 phase of the cell cycle, since most of the additional FISH signals were present as single dots and located at interstitial sites of the involved chromosomes.

Relationship between heterochromatic interstitial telomeric sequences and chromosome damage induced by the radiomimetic compound streptonigrin in Chinese hamster ovary cells.

The relationship between (heterochromatic) interstitial telomeric sequences (ITSs) and the chromosome damage induced by the radiomimetic compound streptonigrin (SN) was investigated in Chinese hamster ovary (CHO) cells by using PNA- and Q-FISH techniques with a pantelomeric probe. CHO cells were exposed to increasing concentrations of SN and chromosomal aberrations were analyzed in the first mitosis after treatment. Cytogenetic analysis revealed that 16.9% and 11.7% of the total aberrations induced by SN in cells harvested 18 h and 3 h after treatment, respectively, exhibited one or more FISH-detectable telomeric signals. Although there was a significant induction by SN of chromosome breaks at centromeric regions containing ITSs, about 70% of the chromosome breaks exhibiting telomeric signals observed in SN-treated cells occurred outside the centromeric regions of chromosomes. This observation, along with the finding of entirely labeled acentric fragments in both untreated and SN-treated cells show that, although this antibiotic induces breakage at centromeric regions containing ITSs, these chromosome regions are not the preferential target for the clastogenic action of SN. In addition, our results show that heterochromatic ITSs are involved more than expected in the formation of chromatid breaks and exchanges induced by SN, and that these sequences are not preferentially involved in the formation of dicentrics, multicentrics, centric rings, chromosome breaks, acentric fragments and chromatid deletions induced by this antibiotic. These findings indicate that the involvement of heterochromatic ITSs in the chromosome damage induced by SN is not random. Moreover, our results show that SN induces telomeric repeats translocations, although this effect depends on the concentration of the drug, and that this antibiotic increases the size of ITSs, this latter effect not being related to the chromosomal sensitivity of the exposed cells to this compound. The mechanism by which SN induces amplification of heterochromatic ITSs remains to be elucidated.

Effect of bleomycin on interstitial telomeric sequences of immortalized Chinese hamster ovary cells.

The effect of the radiomimetic compound bleomycin (BLM) on interstitial telomeric sequences (ITSs) was investigated in Chinese hamster ovary (CHO) cells by using PNA-FISH with a pantelomeric probe. CHO cells were exposed to increasing concentrations of BLM and chromosomal aberrations were analyzed in the first mitosis after treatment. Cytogenetic analysis revealed that 18.1% and 9.5% of the total aberrations observed in cells exposed to BLM and harvested 18h and 3h after treatment, respectively, exhibited one or more FISH-detectable telomeric signals. Most of the chromosome breaks exhibiting telomeric signals observed in BLM-treated cells occurred in the centromeric regions of chromosomes. This observation, along with the finding of entirely labeled acentric fragments in BLM-exposed cells but not in untreated cells, shows that this antibiotic induces breakage at chromosomal sites containing ITSs. In addition, our results show that heterochromatic ITSs are involved more than expected in the formation of chromosome/chromatid breaks - and perhaps chromatid exchanges - induced by BLM, taking into account the percentage of the genome covered by telomeric sequences. On the contrary, our data strongly suggest that ITSs are not preferentially involved in the formation of dicentrics, multicentrics, centric rings, acentric fragments or chromatid deletions induced by BLM. Moreover, our results show that BLM is capable of inducing amplification and translocation of telomeric repeats, and suggest that this antibiotic produces breakage within centromeric ITSs, although chromosome regions containing these sequences are not the preferential target for BLM clastogenic action. On the other hand, our results show that BLM treatment increases the size of ITSs and that this effect is not related to the chromosomal sensitivity of the exposed cells to this compound.

Analysis of spontaneous and bleomycin-induced chromosome damage in peripheral lymphocytes of long-haul aircrew members from Argentina.

Spontaneous and bleomycin (BLM)-induced chromosomal aberrations in G0 and G2 stages of the cell cycle have been analyzed in peripheral lymphocytes of 21 long-haul aircrew members from Argentina in order to assess BLM-induced clastogenesis as a first approach to determine the DNA repair capacity and thereby the susceptibility to environmental cancers in aircrew. The possibility that occupational exposure of flight personnel to cosmic radiation can induce an adaptive response in their peripheral lymphocytes that can be detected by a subsequent in vitro treatment with BLM was also investigated. For comparison, aberrations were also scored in the lymphocytes of 15 healthy volunteers matched by age, health, sex, drinking and smoking habits to the flight personnel group. Aircrew exhibited a higher frequency of spontaneous dicentrics and ring chromosomes than the control population (p<0.05). BLM sensitivity test showed that aircrew and controls are equally sensitive to BLM G2 clastogenic effects, since both groups exhibited a similar frequency of chromatid breaks per cell (p>0.05). However, the aircrew sampled population was almost two times more sensitive to BLM G0 clastogenic effects than controls (p<0.05). Therefore, our data suggest that chronic exposure of aircrew to cosmic radiation increases the in vitro chromosomal sensitivity of their peripheral lymphocytes to BLM (at least in the G0 stage of the cell cycle), and that occupational exposure of flight personnel to cosmic radiation does not induce an adaptive response to this radiomimetic compound. Our results justify further studies aimed at determine if those aircrew members hypersensitive to BLM are more prone to develop environmental cancer than BLM-insensitive individuals.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.