Chromosomal aberrations in PARP−/− mice: Genome stabilization in immortalized cells by reintroduction of poly(ADP-ribose) polymerase cDNA

Depletion of poly(ADP-ribose) polymerase (PARP) increases the frequency of recombination, gene amplification, sister chromatid exchanges, and micronuclei formation in cells exposed to genotoxic agents, implicating PARP in the maintenance of genomic stability. Flow cytometric analysis now has revealed an unstable tetraploid population in immortalized fibroblasts derived from PARP−/− mice. Comparative genomic hybridization detected partial chromosomal gains in 4C5-ter, 5F-ter, and 14A1-C1 in PARP−/−mice and immortalized PARP−/−fibroblasts. Neither the chromosomal gains nor the tetraploid population were apparent in PARP−/− cells stably transfected with PARP cDNA [PARP−/−(+PARP)], indicating negative selection of cells with these genetic aberrations after reintroduction of PARP cDNA. Although the tumor suppressor p53 was not detectable in PARP−/− cells, p53 expression was partially restored in PARP−/− (+PARP) cells. Loss of 14D3-ter that encompasses the tumor suppressor gene Rb-1 in PARP−/− mice was associated with a reduction in retinoblastoma(Rb) expression; increased expression of the oncogene Jun was correlated with a gain in 4C5-ter that harbors this oncogene. These results further implicate PARP in the maintenance of genomic stability and suggest that altered expression of p53, Rb, and Jun, as well as undoubtedly many other proteins may be a result of genomic instability associated with PARP deficiency.

Etoposide induces heritable chromosomal aberrations and aneuploidy during male meiosis in the mouse

Etoposide, a topoisomerase II inhibitor widely used in cancer therapy, is suspected of inducing secondary tumors and affecting the genetic constitution of germ cells. A better understanding of the potential heritable risk of etoposide is needed to provide sound genetic counseling to cancer patients treated with this drug in their reproductive years. We used a mouse model to investigate the effects of clinical doses of etoposide on the induction of chromosomal abnormalities in spermatocytes and their transmission to zygotes by using a combination of chromosome painting and 4′,6-diamidino-2-phenylindole staining. High frequencies of chromosomal aberrations were detected in spermatocytes within 64 h after treatment when over 30% of the metaphases analyzed had structural aberrations (P < 0.01). Significant increases in the percentages of zygotic metaphases with structural aberrations were found only for matings that sampled treated pachytene (28-fold, P < 0.0001) and preleptotene spermatocytes (13-fold, P < 0.001). Etoposide induced mostly acentric fragments and deletions, types of aberrations expected to result in embryonic lethality, because they represent loss of genetic material. Chromosomal exchanges were rare. Etoposide treatment of pachytene cells induced aneuploidy in both spermatocytes (18-fold, P < 0.01) and zygotes (8-fold, P < 0.05). We know of no other report of an agent for which paternal exposure leads to an increased incidence of aneuploidy in the offspring. Thus, we found that therapeutic doses of etoposide affect primarily meiotic germ cells, producing unstable structural aberrations and aneuploidy, effects that are transmitted to the progeny. This finding suggests that individuals who undergo chemotherapy with etoposide may be at a higher risk for abnormal reproductive outcomes especially within the 2 months after chemotherapy.

The human XRCC9 gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells

The Chinese hamster ovary (CHO) mutant UV40 cell line is hypersensitive to UV and ionizing radiation, simple alkylating agents, and DNA cross-linking agents. The mutant cells also have a high level of spontaneous chromosomal aberrations and 3-fold elevated sister chromatid exchange. We cloned and sequenced a human cDNA, designated XRCC9, that partially corrected the hypersensitivity of UV40 to mitomycin C, cisplatin, ethyl methanesulfonate, UV, and γ-radiation. The spontaneous chromosomal aberrations in XRCC9 cDNA transformants were almost fully corrected whereas sister chromatid exchanges were unchanged. The XRCC9 genomic sequence was cloned and mapped to chromosome 9p13. The translated XRCC9 sequence of 622 amino acids has no similarity with known proteins. The 2.5-kb XRCC9 mRNA seen in the parental cells was undetectable in UV40 cells. The mRNA levels in testis were up to 10-fold higher compared with other human tissues and up to 100-fold higher compared with other baboon tissues. XRCC9 is a candidate tumor suppressor gene that might operate in a postreplication repair or a cell cycle checkpoint function.

Perturbation of the T cell repertoire in rheumatoid arthritis

Aberrations in the T cell repertoire with the emergence of oligoclonal populations have been described in patients with rheumatoid arthritis (RA). However, the extent of the repertoire perturbations as well as the underlying mechanisms are not known. We now have examined the diversity of the peripheral CD4 T cell repertoire by determining the frequencies of arbitrarily selected T cell receptor (TCR) β-chain sequences. Healthy individuals displayed a highly diverse repertoire, with a median frequency of individual TCR β-chain sequences of 1 in 2.4 × 107 CD4 T cells. In RA patients, the median TCR β-chain frequency was increased 10-fold, indicating marked contraction of the repertoire (P < 0.001). The loss in TCR diversity was not limited to CD4 memory T cells but also involved the compartment of naive T cells, suggesting that it reflected an abnormality in T cell repertoire formation and not a consequence of antigen recognition in the synovium. Also, control patients with chronic inflammatory disease such as hepatitis C expressed a diverse repertoire indistinguishable from that of normals. Telomere length studies indicated an increased replicative history of peripheral CD4 T cells in RA patients, suggesting an enhanced turnover within the CD4 compartment. Compared with age-matched controls, terminal restriction fragment sizes were 1.7 kilobases shorter (P < 0.001). These data demonstrate an altered CD4 T cell homeostasis in RA that may contribute to the autoimmune response as well as to the immunodeficiency in these patients.

A homeobox gene with potential developmental control function in the meristem of the conifer Picea abies

Many homeobox genes control essential developmental processes in animals and plants. In this report, we describe the first cDNA corresponding to a homeobox gene isolated from a gymnosperm, the HBK1 gene from the conifer Picea abies (L.) Karst (Norway spruce). The sequence shows distinct similarities specifically to the KNOX (knotted-like homeobox) class of homeobox genes known from different angiosperm plants. The deduced amino acid sequence of HBK1 is strikingly similar within the homeodomain (84% identical) to the maize gene Knotted1 (Kn1), which acts to regulate cell differentiation in the shoot meristem. This similarity suggested that the phylogenetic association of HBK1 with the KNOX genes might be coupled to a conservation of gene function. In support of this suggestion, we have found HBK1 to be expressed in the apical meristem in the central population of nondifferentiated stem cells, but not in organ primordia developing at the flanks of the meristem. This pattern of expression is similar to that of Kn1 in the maize meristem. We show further that HBK1, when expressed ectopically in transgenic Arabidopsis plants, causes aberrations in leaf development that are similar to the effects of ectopic expression of angiosperm KNOX genes on Arabidopsis development. Taken together, these data suggest that HBK1 has a role, similar to the KNOX genes in angiosperms, in the control of cellular differentiation in the apical meristem of spruce. The data also indicate that KNOX-gene regulation of vegetative development is an ancient feature of seed plants that was present in the last common ancestor of conifers and angiosperms.

DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes

Recent findings intriguingly place DNA double-strand break repair proteins at chromosome ends in yeast, where they help maintain normal telomere length and structure. In the present study, an essential telomere function, the ability to cap and thereby protect chromosomes from end-to-end fusions, was assessed in repair-deficient mouse cell lines. By using fluorescence in situ hybridization with a probe to telomeric DNA, spontaneously occurring chromosome aberrations were examined for telomere signal at the points of fusion, a clear indication of impaired end-capping. Telomeric fusions were not observed in any of the repair-proficient controls and occurred only rarely in a p53 null mutant. In striking contrast, chromosomal end fusions that retained telomeric sequence were observed in nontransformed DNA-PKcs-deficient cells, where they were a major source of chromosomal instability. Metacentric chromosomes created by telomeric fusion became even more abundant in these cells after spontaneous immortalization. Restoration of repair proficiency through transfection with a functional cDNA copy of the human DNA-PKcs gene reduced the number of fusions compared with a negative transfection control. Virally transformed cells derived from Ku70 and Ku80 knockout mice also displayed end-to-end fusions. These studies demonstrate that DNA double-strand break repair genes play a dual role in maintaining chromosomal stability in mammalian cells, the known role in repairing incidental DNA damage, as well as a new protective role in telomeric end-capping.

Defects in transforming growth factor-β signaling cooperate with a Ras oncogene to cause rapid aneuploidy and malignant transformation of mouse keratinocytes

Genetic inactivation of the transforming growth factor-β (TGF-β) signaling pathway can accelerate tumor progression in the mouse epidermal model of multistage carcinogenesis. By using an in vitro model of keratinocyte transformation that parallels in vivo malignant conversion to squamous cell carcinoma, we show that v-rasHa transduced primary TGF-β1−/− keratinocytes and keratinocytes expressing a TGF-β type II dominant-negative receptor transgene have significantly higher frequencies of spontaneous transformation than control genotypes. Malignant transformation in the TGF-β1−/− keratinocytes is preceded by aneuploidy and accumulation of chromosomal aberrations. Similarly, transient inactivation of TGF-β signaling with a type II dominant-negative receptor adenovirus causes rapid changes in ploidy. Exogenous TGF-β1 can suppress aneuploidy, chromosome breaks, and malignant transformation of the TGF-β1−/− keratinocytes at concentrations that do not significantly arrest cell proliferation. These results point to genomic instability as a mechanism by which defects in TGF-β signaling could accelerate tumor progression in mouse multistage carcinogenesis.

Analysis of genomic alterations in benign, atypical, and anaplastic meningiomas: Toward a genetic model of meningioma progression

Nineteen benign [World Health Organization (WHO) grade I; MI], 21 atypical (WHO grade II; MII), and 19 anaplastic (WHO grade III; MIII) sporadic meningiomas were screened for chromosomal imbalances by comparative genomic hybridization (CGH). These data were supplemented by molecular genetic analyses of selected chromosomal regions and genes. With increasing malignancy grade, a marked accumulation of genomic aberrations was observed; i.e., the numbers (mean ± SEM) of total alterations detected per tumor were 2.9 ± 0.7 for MI, 9.2 ± 1.2 for MII, and 13.3 ± 1.9 for MIII. The most frequent alteration detected in MI was loss on 22q (58%). In MII, aberrations most commonly identified were losses on 1p (76%), 22q (71%), 14q (43%), 18q (43%), 10 (38%), and 6q (33%), as well as gains on 20q (48%), 12q (43%), 15q (43%), 1q (33%), 9q (33%), and 17q (33%). In MIII, most of these alterations were found at similar frequencies. However, an increase in losses on 6q (53%), 10 (68%), and 14q (63%) was observed. In addition, 32% of MIII demonstrated loss on 9p. Homozygous deletions in the CDKN2A gene at 9p21 were found in 4 of 16 MIII (25%). Highly amplified DNA sequences were mapped to 12q13–q15 by CGH in 1 MII. Southern blot analysis of this tumor revealed amplification of CDK4 and MDM2. By CGH, DNA sequences from 17q were found to be amplified in 1 MII and 8 MIII, involving 17q23 in all cases. Despite the high frequency of chromosomal aberrations in the MII and MIII investigated, none of these tumors showed mutations in exons 5–8 of the TP53 gene. On the basis of the most common aberrations identified in the various malignancy grades, a model for the genomic alterations associated with meningioma progression is proposed.

Chromosomal breakage-fusion-bridge events cause genetic intratumor heterogeneity

It has long been known that rearrangements of chromosomes through breakage-fusion-bridge (BFB) cycles may cause variability of phenotypic and genetic traits within a cell population. Because intercellular heterogeneity is often found in neoplastic tissues, we investigated the occurrence of BFB events in human solid tumors. Evidence of frequent BFB events was found in malignancies that showed unspecific chromosome aberrations, including ring chromosomes, dicentric chromosomes, and telomeric associations, as well as extensive intratumor heterogeneity in the pattern of structural changes but not in tumors with tumor-specific aberrations and low variability. Fluorescence in situ hybridization analysis demonstrated that chromosomes participating in anaphase bridge formation were involved in a significantly higher number of structural aberrations than other chromosomes. Tumors with BFB events showed a decreased elimination rate of unstable chromosome aberrations after irradiation compared with normal cells and other tumor cells. This result suggests that a combination of mitotically unstable chromosomes and an elevated tolerance to chromosomal damage leads to constant genomic reorganization in many malignancies, thereby providing a flexible genetic system for clonal evolution and progression.

The IgG Fc receptor, FcγRIIB, is a target for deregulation by chromosomal translocation in malignant lymphoma

Rearrangement of chromosomal bands 1q21–23 is one of the most frequent chromosomal aberrations observed in hematological malignancy. The genes affected by these rearrangements remain poorly characterized. Typically, 1q21–23 rearrangements arise during tumor evolution and accompany disease-specific chromosomal rearrangements such as t(14;18) (BCL2) and t(8;14) (MYC), where they are thus thought to play an important role in tumor progression. The pathogenetic basis of this 1q21–23-associated disease progression is currently unknown. In this setting, we surveyed our series of follicular lymphoma for evidence of recurring 1q21–23 breaks and identified three cases in which a t(14;18)(q32;q21) was accompanied by a novel balanced t(1;22)(q22;q11). Molecular cloning of the t(1;22) in a cell line (B593) derived from one of these cases and detailed fluorescent in situ hybridization mapping in the two remaining cases identified the FCGR2B gene, which encodes the immunoreceptor tyrosine-based inhibition motif-bearing IgG Fc receptor, FcγRIIB, as the target gene of the t(1;22)(q22;q11). We demonstrate deregulation of FCGR2B leading to hyperexpression of FcγRIIb2 as the principal consequence of the t(1;22). This is evidence that IgG Fc receptors can be targets for deregulation through chromosomal translocation in lymphoma. It suggests that dysregulation of FCGR2B may play a role in tumor progression in follicular lymphoma.

Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice

A physiological examination of mice harboring a null allele at the aryl hydrocarbon (Ah) locus revealed that the encoded aryl hydrocarbon receptor plays a role in the resolution of fetal vascular structures during development. Although the aryl hydrocarbon receptor is more commonly studied for its role in regulating xenobiotic metabolism and dioxin toxicity, a developmental role of this protein is supported by the observation that Ah null mice display smaller livers, reduced fecundity, and decreased body weights. Upon investigating the liver phenotype, we found that the decrease in liver size is directly related to a reduction in hepatocyte size. We also found that smaller hepatocyte size is the result of massive portosystemic shunting in null animals. Colloidal carbon uptake and microsphere perfusion studies indicated that 56% of portal blood flow bypasses the liver sinusoids. Latex corrosion casts and angiography demonstrated that shunting is consistent with the existence of a patent ductus venosus in adult animals. Importantly, fetal vascular structures were also observed at other sites. Intravital microscopy demonstrated an immature sinusoidal architecture in the liver and persistent hyaloid arteries in the eyes of adult Ah null mice, whereas corrosion casting experiments described aberrations in kidney vascular patterns.

Spectral karyotyping suggests additional subsets of colorectal cancers characterized by pattern of chromosome rearrangement

The abundant chromosome abnormalities in most carcinomas are probably a reflection of genomic instability present in the tumor, so the pattern and variability of chromosome abnormalities will reflect the mechanism of instability combined with the effects of selection. Chromosome rearrangement was investigated in 17 colorectal carcinoma-derived cell lines. Comparative genomic hybridization showed that the chromosome changes were representative of those found in primary tumors. Spectral karyotyping (SKY) showed that translocations were very varied and mostly unbalanced, with no translocation occurring in more than three lines. At least three karyotype patterns could be distinguished. Some lines had few chromosome abnormalities: they all showed microsatellite instability, the replication error (RER)+ phenotype. Most lines had many chromosome abnormalities: at least seven showed a surprisingly consistent pattern, characterized by multiple unbalanced translocations and intermetaphase variation, with chromosome numbers around triploid, 6–16 structural aberrations, and similarities in gains and losses. Almost all of these were RER−, but one, LS411, was RER+. The line HCA7 showed a novel pattern, suggesting a third kind of genomic instability: multiple reciprocal translocations, with little numerical change or variability. This line was also RER+. The coexistence in one tumor of two kinds of genomic instability is to be expected if the underlying defects are selected for in tumor evolution.

Computational adaptive optics for live three-dimensional biological imaging

Light microscopy of thick biological samples, such as tissues, is often limited by aberrations caused by refractive index variations within the sample itself. This problem is particularly severe for live imaging, a field of great current excitement due to the development of inherently fluorescent proteins. We describe a method of removing such aberrations computationally by mapping the refractive index of the sample using differential interference contrast microscopy, modeling the aberrations by ray tracing through this index map, and using space-variant deconvolution to remove aberrations. This approach will open possibilities to study weakly labeled molecules in difficult-to-image live specimens.

Direct toxicity of nonsteroidal antiinflammatory drugs for renal medullary cells

Antipyretic analgesics, taken in large doses over a prolonged period, cause a specific form of kidney disease, characterized by papillary necrosis and interstitial scarring. Epidemiological evidence incriminated mixtures of drugs including aspirin (ASA), phenacetin, and caffeine. The mechanism of toxicity is unclear. We tested the effects of ASA, acetaminophen (APAF, the active metabolite of phenacetin), caffeine, and other related drugs individually and in combination on mouse inner medullary collecting duct cells (mIMCD3). The number of rapidly proliferating cells was reduced by ≈50% by 0.5 mM ASA, salicylic acid, or APAF. The drugs had less effect on confluent cells, which proliferate slowly. Thus, the slow in vivo turnover of IMCD cells could explain why clinical toxicity requires very high doses of these drugs over a very long period. Caffeine greatly potentiated the effect of acetaminophen, pointing to a potential danger of the mixture. Cyclooxygenase (COX) inhibitors, indomethacin and NS-398, did not reduce cell number except at concentrations greatly in excess of those that inhibit COX. Therefore, COX inhibition alone is not toxic. APAF arrests most cells in late G1 and S and produces a mixed form of cell death with both oncosis (swollen cells and nuclei) and apoptosis. APAF is known to inhibit the synthesis of DNA and cause chromosomal aberrations due to inhibition of ribonucleotide reductase. Such effects of APAF might account for renal medullary cell death in vivo and development of uroepithelial tumors from surviving cells that have chromosomal aberrations.

UV-induced Hyperphosphorylation of Replication Protein A Depends on DNA Replication and Expression of ATM Protein

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4–8 h) to UV radiation (10–30 J/m2). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.