Laura Gómez

Effect of hydroxyurea and normal plasma on DNA synthesis in lymphocytes from Fanconi anemia patients.

Fanconi anemia (FA) is characterized at the cellular level by a high frequency of spontaneous chromosomal aberrations; crosslinking agents cause an abnormal increase in the frequency of chromosomal damage, and semiconservative DNA synthesis is severely inhibited. Deoxyribonucleotides are needed in both semiconservative and repair DNA synthesis. To investigate the involvement of deoxyribonucleotide pools in the inhibition of DNA synthesis in FA, we evaluated the effect on FA lymphocytes of hydroxyurea (HU), an inhibitor of ribonucleotide reductase which is known to alter the intracellular levels of deoxyribonucleotides. To achieve this goal, lymphocyte cultures of 4 FA patients and 4 normal individuals were used. Cultures were treated with HU and/or mitomycin C and normal human plasma. All cultures were processed to detect the number of DNA synthesizing nuclei by autoradiography. Scoring of 2000 nuclei for each kind of culture every 6 h in the last 24 h of incubation showed that, in long incubation periods, DNA synthesis in FA is largely inhibited by HU and this hypersensitivity may be partially decreased by addition of normal human plasma. It is known that recovery from damage induced by HU involves several enzymes such as flavin oxido-reductase, superoxide dismutase and catalase which are involved in the production or scavenging of O2 radicals; FA cells are deficient in the detoxification of oxygen and this could explain the response of FA cells to HU.

Chromosome instability with bleomycin and x‐ray hypersensitivity in a boy with Nijmegen breakage syndrome

We report on a Mexican boy with microcephaly, short stature, and a high frequency of chromosome aberrations with rearrangements involving chromosomes 7 and 14, typical of ataxia telangiectasia (AT) patients. He had neither ataxia nor telangiectasia, and his immunological status and serum alpha feto protein (AFP) level were normal. Bleomycin hypersensitivity, which has been demon-strated in AT patients, was tested in the patient using AT and normal subjects for comparison. The frequency of spontaneously occurring chromosome aberrations in lymphocyte cultures was significantly higher in the patient and the AT patient than in the normal subject. Four cells from the patient showed structural rearrangements involving chromosomes 7 or 14, with breakpoints typical for AT. When exposed to 5.0 micrograms bleomycin, the lymphocytes from the AT patient showed the highest sensitivity to this agent; our patient had an intermediate sensitivity. In both patients several rearrangements involving chromosomes 7 and 14 were scored, while none were observed in the normal subject. A colony survival assay (CSA) [Huo et al., 1994: Cancer Res 54:2544-2547], using a lymphoblastoid cell line (LCL) derived from our patient, showed a survival fraction (SF) of 7%, which is in the same range as in AT patients. The clinical picture, together with the cytogenetic and radiosensitivity results, suggests that our patient fits the variable spectrum of Nijmegen breakage syndrome.

Differential expression of TP53 associated genes in Fanconi anemia cells after mitomycin C and hydroxyurea treatment

Fanconi anemia (FA) is a rare, heritable chromosomal instability disease characterized by several congenital defects and cancer predisposition. Functional interactions between specific FA proteins and DNA damage response and repair activities have been reported, but the interplay between these mechanisms for maintaining genomic stability are not well understood. Many DNA damage response proteins are transcriptionally regulated by the tumor suppressor protein p53 (TP53), suggesting an important regulatory role for the DNA damage and stress response pathway. To better understand the association between FA and the DNA damage stress response we analyzed the levels of chromosomal damage and damage mediated gene transcription responses in lymphoblastoid cells derived from normal individuals and patients carrying the most common FA complementation group (FA-A). Chromosomal aberrations were first measured after exposure to mitomicyn C (MMC) or hydroxyurea (HU). Aliquots of the same cell were than assayed for the transcriptional response of 21 DNA damage and stress response genes using quantitative real-time PCR. The FA-A lymphoblastoid cells showed significant increases in the frequency of chromosome aberrations relative to non-FA-A lymphoblastoid lines after MMC treatment. The MMC induced damage was correlated with a general increase in expression of TP53-modulated DNA damage stress response genes involved in processes such as DNA repair, cell cycle progression, and apoptosis. Following HU treatment FA cells showed a decreased induction of CAs with much less transcriptional differences between targeted genes. Overall, the differences between the normal and FA-A cells after genotoxic treatments imply an increased activation and reliance of FA cells on the down-stream activities of TP53 for prevention of cell killing and chromosome damage from interstrand crosslinks but not for general replication arrest and double strand breaks. Furthermore, these results imply a regulatory connection between the FA pathway and activation of TP53 for responding to DNA damage. Alterations in the regulation of the DNA damage response may be related to the complex phenotypes seen in FA patients.

Ataxia-pancytopenia syndrome

We report on a Mexican girl who developed cerebellar ataxia at age 3 years and pancytopenia at age 13 years. Cerebral computed tomography scan and magnetic resonance imaging showed evidence of severe cerebellar atrophy. Telangiectasias were not present; immunoglobulins and alpha-fetoprotein levels were normal. Cytogenetic studies showed no evidence of spontaneous chromosome aberrations, a normal rate of diepoxybutane (DEB) and mitomycin C (MMC)-induced chromosome aberrations, but an increased response to bleomycin. The phenotype support the diagnosis of ataxia-pancytopenia syndrome, although monosomy of chromosome 7 was not found in bone marrow. The cytogenetic studies suggest that this may be a chromosomal instability disorder.

Hydroxyurea induces chromosomal damage in G2 and enhances the clastogenic effect of mitomycin C in Fanconi anemia cells

Fanconis anemia (FA) is a recessive disease; 16 genes are currently recognized in FA. FA proteins participate in the FA/BRCA pathway that plays a crucial role in the repair of DNA damage induced by crosslinking compounds. Hydroxyurea (HU) is an agent that induces replicative stress by inhibiting ribonucleotide reductase (RNR), which synthesizes deoxyribonucleotide triphosphates (dNTPs) necessary for DNA replication and repair. HU is known to activate the FA pathway; however, its clastogenic effects are not well characterized. We have investigated the effects of HU treatment alone or in sequential combination with mitomycin‐C (MMC) on FA patient‐derived lymphoblastoid cell lines from groups FA‐A, B, C, D1/BRCA2, and E and on lymphocytes from two unclassified FA patients. All FA cells showed a significant increase (P < 0.05) in chromosomal aberrations following treatment with HU during the last 3 h before mitosis. Furthermore, when FA cells previously exposed to MMC were treated with HU, we observed an increase of MMC‐induced DNA damage that was characterized by high occurrence of DNA breaks and a reduction in rejoined chromosomal aberrations. These findings show that exposure to HU during G2 induces chromosomal aberrations by a mechanism that is independent of its well‐known role in replication fork stalling during S‐phase and that HU interfered mainly with the rejoining process of DNA damage. We suggest that impaired oxidative stress response, lack of an adequate amount of dNTPs for DNA repair due to RNR inhibition, and interference with cell cycle control checkpoints underlie the clastogenic activity of HU in FA cells. Environ. Mol. Mutagen. 56:457–467, 2015.