B NBS Cellular Phenotypes

1. Radiosensitivity

The documented clinical radiosensitivity of patients with NBS is reflected by at least a two-fold increase in cellular hypersensitivity to ionizing radiation (IR) of primary and immortalized NBS cells (2,10). This hypersensitivity is also evident following exposure of NBS cells to bleomycin, streptonigrin, etoposide, camp-tothecin, and the cross-linking agent mitomycin C, but not to ultraviolet wavelength C (UV-C) (32-34), indicating that DNA double-strand breaks (DSBs), produced through different mechanisms by these agents, are the crucial molecular lesions for cell killing in NBS. However, the mechanism leading to NBS cell death following induction of DSBs has not been determined (e.g., no evidence for increased apoptosis has been described). Indeed, one report even showed diminished IR-induced apoptosis in leukocytes from a patient with NBS (35).

Hypersensitivity to DSB-producing agents and the concomitant observation of increased spontaneous and IR-induced chromosomal aberrations resulted early on in the concept that NBS cells are deficient in either repair and/or signaling of DSB-damaged genomic DNA or DNA-damage-dependent cell cycle regulation.

2. Cell Cycle Checkpoint Deficiencies

Following the induction of DSBs by IR, replicating cells are transiently arrested in cell cycle progression upon reaching cell cycle checkpoints in the G1, S, or G2 phase. It is widely believed that the transient cell cycle arrest serves to repair the DSBs prior to replication of damaged DNA templates and mitosis. Thus, cell cycle checkpoints may play crucial roles in determining the fate of damaged cells, as is exemplified in certain yeast mutants (36). In accordance with this hypothesis, cell cycle checkpoint deficiencies in G1, S, and G2 have been demonstrated conclusively in radiosensitive AT cells. The G1 arrest following IR is mainly dependent on the ataxia-telangiectasia mutated (ATM)/p53 pathway (37). Upon IR, p53 is phosphorylated by the kinase activity of ATM and thus stabilized in the G1 phase. Accumulation of the p53 protein, in turn, induces p21 transcription and subsequent G1 arrest. Several studies have shown that, similar to AT cells, NBS cells exhibit delayed and reduced accumulation of p53 and reduced p53-depen-dent activation of p21 and other genes in response to IR (38-42). However, although the accumulation of p53 is clearly blunted following IR, a normal G1 arrest has been observed in all NBS fibroblast lines tested (40,42). In contrast, the G1 arrest was defective in one NBS lymphoblastoid line (33) and intermediate among normal and AT cells in other lymphoblastoid NBS cell lines (41).

Inhibition of DNA replication (i.e., the S phase arrest) is a very fast response in actively replicating cells treated with IR (43,44). This decrease in the rate of DNA synthesis typically follows a biphasic kinetic curve, with a first steep component that presumably reflects blockage of initiation by preventing firing at origins of replication, and a second shallow component due to inhibition of chain elongation. In NBS cells, inhibition of DNA synthesis following IR is greatly reduced, a phenomenon termed radioresistant DNA synthesis (RDS) (45,46). RDS has been observed in virtually all NBS cell lines tested so far (for one exception, see ref. 42) and is a distinctive phenotype of cells from patients with NBS, AT, and ataxia-telangiectasia-like disorder (ATLD)—the latter being attributable to mutations in the hMRE11 gene (47). Molecular analyses have shown that besides NBS1, hMRE11, and ATM, other genes may be involved in the regulation of RDS, such as an unknown gene on human chromosome 4 (48), and components of a calmodulin-dependent regulatory cascade (49). Recovery from radiation-induced DNA synthesis inhibition is supported by the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) but not Ku80, DNA ligase 5, or XRCC4 (50), illustrating the high complexity of pathways controlling DNA synthesis in response to IR.

As for the G1 arrest, data on IR-induced G2 arrest are somewhat controversial. Whereas Yamazaki (40) and Girard (42) found no obvious defect in IR-induced G2 arrest in NBS fibroblasts, Ito (51) has described a prolonged G2 phase compared with control cells. Antoccia (52) also has found differences in the IR-induced G2 arrest among NBS and AT as well as normal lymphoblastoid cell lines.

There may be several reasons for the inconsistencies in experimental data relating to the IR-induced G1 and G2 arrest in NBS cells. Different experimental results may depend on the particular cell type used, on their different genetic background, with respect to the NBS1 or secondary modifying mutations, as well as on different experimental setups. Clearly, however, RDS is a major hallmark and key diagnostic feature of the cellular NBS phenotype.

3. Genomic Instability

Spontaneous chromosomal instability in cells from patients with NBS is evident in T lymphocytes, with frequent rearrangements being observed at the loci of im-munoglobulin and T-cell receptor genes on chromosomes 7 and 14 (2,9). In cultured (and immortalized) NBS lymphoblast and fibroblast cell lines, spontaneous chromosomal instability is characterized mainly by an elevated frequency of chromatid breaks occurring at random sites (41,42). In addition, telomeric fusions leading to dicentric chromosomes have been observed with increased frequency in metaphases from NBS cells.

Following irradiation in the G1 phase, the frequency of IR-induced aberrations is drastically increased in cultured NBS cells compared with normal cells. For example, following 2-Gy IR, the frequency of total chromosomal aberrations shows a 2.4- to 3.9-fold increase in NBS cells as compared with normal cells (41). Interestingly, the spectrum of aberration types is different in NBS. Whereas normal cells display mainly chromosome-type aberrations following IR, metaphases from NBS cells are characterized by up to an 8- to 13-fold higher amount of chro-matid-type aberrations compared with normal cells (17,41,45,52,53). Concomitant with the increased aberration frequencies, the percentage of damaged metaphases also is increased about two fold in NBS versus normal cells (41).

4. DNA Repair Defect

In contrast to the obviously increased numbers of spontaneous and IR-induced chromosomal aberrations, using pulsed-field gel electrophoresis (PFGE) demonstrates no (34,54), or at best very little (42), deficiency in NBS cells regarding the kinetics and efficiency of DSB rejoining following high doses of IR. The same holds true for AT cells.

Premature chromosomal condensation (PCC) has been used to detect nonrepaired DNA damage that leads to chromosomal breaks. In this assay, two primary NBS fibroblast lines showed slightly elevated frequencies of extrachromosomal fragments 24 hrs following IR compared with normal cells, indicating aberrant repair in NBS (42). In addition, the formation of micronuclei that arises when acentric fragments are generated was found to be slightly higher in untreated NBS (as well as AT) cells compared with normal cells. Following IR, hypersensitivity in micronuclei formation was significant only at higher doses (42). It has been reported that dinucleotide repeat sequences are stable over time in NBS cells, indicative of normal DNA mismatch repair systems and normal replicative fidelity in NBS (33). Also, normal DNA-PKcs and Ku levels, as well as normal DNA-PKcs activity in vitro have been reported in NBS protein extracts (33), suggesting that this pathway of DNA repair was normal in cells from a NBS patient.

Collectively, the data reflect an obvious inconsistency between apparently normal, or at best slightly deficient, DNA DSB repair and high levels of chromosomal aberrations in NBS. This may be due to limitations in the method used, since small numbers of nonrepaired DSBs, almost undetectable in PFGE, ultimately may lead to chromosomal aberrations and resultant IR hypersensitivity. In this regard, it has been reported that few or even one unrepaired DSB may be lethal for an individual cell (55). Alternatively, this disparity may reflect the possibility that the efficiency of DSB rejoining, per se, is not affected in NBS but rather the fidelity of DSB joining may be error prone. Finally, multiple independent pathways of nonhomologous DSB repair may have redundant and overlapping functions, thus masking the deficiency of the NBS1 pathway in terms of DSB-rejoining efficiency.

5. Phenotypic Correlations

Although cell cycle deficiency is expected to play a causative role in chromosomal instability and radiation sensitivity, recent experimental data suggest that cell cycle checkpoints may not be a crucial determinant of this phenotype in AT (56-58) and NBS. Girard et al. (42) have demonstrated that primary fibroblasts from different patients with NBS, all of which share the same common founder mutation, display different RDS deficiencies and IR-induced cell cycle checkpoints, while retaining the hypersensitivity to IR regarding cell survival and DNA repair assays. Moreover, an increase in chromosomal aberrations and decreased DNA repair following IR have been demonstrated in noncycling cells, thus ruling out the possibility that cell cycle deficiencies may contribute to IR-sensitive phe-notypes. Likewise, it has been demonstrated that the RDS phenotype is not solely responsible for IR sensitivity and chromosomal instability in NBS and AT cells (48,57). Some investigators therefore have suggested that despite their limited detection, defects in DNA repair rather than in cell cycle checkpoints may be the primary reason for IR sensitivity in NBS and AT cells (56).

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