Rapid quasi-periodic oscillations in the relativistic jet of BL Lacertae.

Blazars are active galactic nuclei (AGN) with relativistic jets whose non-thermal radiation is extremely variable on various timescales1-3. This variability seems mostly random, although some quasi-periodic oscillations (QPOs), implying systematic processes, have been reported in blazars and other AGN. QPOs with timescales of days or hours are especially rare4 in AGN and their nature is highly debated, explained by emitting plasma moving helically inside the jet5, plasma instabilities6,7 or orbital motion in an accretion disc7,8. Here we report results of intense optical and γ-ray flux monitoring of BL Lacertae (BL Lac) during a dramatic outburst in 2020 (ref. 9). BL Lac, the prototype of a subclass of blazars10, is powered by a 1.7 × 108 MSun (ref. 11) black hole in an elliptical galaxy (distance = 313 megaparsecs (ref. 12)). Our observations show QPOs of optical flux and linear polarization, and γ-ray flux, with cycles as short as approximately 13 h during the highest state of the outburst. The QPO properties match the expectations of current-driven kink instabilities6 near a recollimation shock about 5 parsecs (pc) from the black hole in the wake of an apparent superluminal feature moving down the jet. Such a kink is apparent in a microwave Very Long Baseline Array (VLBA) image.

Ataxia telangiectasia mutated is essential during adult neurogenesis.

Ataxia telangiectasia (A-T) is an autosomal recessive disease characterized by normal brain development followed by progressive neurodegeneration. The gene mutated in A-T (ATM) is a serine protein kinase implicated in cell cycle regulation and DNA repair. The role of ATM in the brain and the consequences of its loss on neuronal survival remain unclear. We studied the role of ATM in adult neural progenitor cells in vivo and in vitro to define the role of ATM in dividing and postmitotic neural cells from Atm-deficient (Atm(-/-)) mice in a physiologic context. We demonstrate that ATM is an abundant protein in dividing neural progenitor cells but is markedly down-regulated as cells differentiate. In the absence of ATM, neural progenitor cells of the dentate gyrus show abnormally high rates of proliferation and genomic instability. Atm(-/-) cells in vivo, and in cell culture, show a blunted response to environmental stimuli that promote neural progenitor cell proliferation, survival, and differentiation along a neuronal lineage. This study defines a role for ATM during the process of neurogenesis, demonstrates that ATM is required for normal cell fate determination and neuronal survival both in vitro and in vivo, and points to a mechanism for neuronal cell loss in progressive neurodegenerative diseases.

Comparative genomic hybridization (CGH)--detection of unbalanced genetic aberrations using conventional and micro-array techniques.

This unit presents comparative genomic hybridization (CGH), a genome-wide screening technique for genetic aberrations in tumor samples. Specific emphasis is placed on recent applications to the analysis of murine model systems for human cancer. CGH is an invaluable tool for identifying the characteristic genetic rearrangements in these models. The authors discuss an exciting new method currently being developed, array CGH, which results in a tremendous increase in resolution. Oncogene amplifications and deletions of tumor-suppressor genes are detected on a single-gene level. Detailed protocols are supplied for CGH analysis of both human and mouse chromosomes.

Abnormal rearrangement within the alpha/delta T-cell receptor locus in lymphomas from Atm-deficient mice.

Atm-deficient mice (Atm(-/-)) recapitulate many aspects of the ataxia telangiectasia (AT) syndrome, including the susceptibility to tumors of lymphoid origin. To investigate the mechanism of tumorigenesis, we have examined a panel of 8 thymic lymphomas from Atm(-/-) mice. All Atm(-/-) tumors are of thymic lymphoblastoid origin, display an immature CD3(-) and CD4(+)/CD8(+) phenotype, and arise coincident with V(D)J recombination. Cytogenetically, all tumors are diploid or near diploid but exhibit multiple chromosome aberrations with an average of 4 abnormal chromosomes per tumor. All the tumors revealed chromosome 14 rearrangements precisely at the T-cell receptoralpha/delta (Tcralpha/delta) locus, suggesting the involvement of V(D)J recombination in these translocations. In addition, 11.5% of Atm(-/-) peripheral T cells showed chromosome 14 translocations, suggesting that rearrangements at the Tcralpha/delta locus occur early during tumor development in the absence of ATM. However, additional genetic aberrations are required for tumorigenesis. For example, translocations involving chromosome 12, often with chromosome 14 (more than 60%), and partial or complete trisomy of chromosome 15, with copy number increases of the c-myc oncogene were frequently observed. These observations suggest that ATM is required for normal rearrangement of the Tcralpha/delta locus but not for V(D)J recombination at other loci. The mechanisms that lead to tumorigenesis may be due to the involvement of ATM in monitoring double-stranded DNA breaks. (Blood. 2000;96:1940-1946)

Recombinase-activating gene (RAG) 2-mediated V(D)J recombination is not essential for tumorigenesis in Atm-deficient mice.

The majority of Atm-deficient mice die of malignant thymic lymphoma by 4-5 mo of age. Cytogenetic abnormalities in these tumors are consistently identified within the Tcr alpha/delta locus, suggesting that tumorigenesis is secondary to aberrant responses to double-stranded DNA breaks that occur during V(D)J recombination. Since V(D)J recombination is a recombinase-activating gene (RAG)-dependent process, we generated Rag2(-/-)Atm(-/-) mice to assess the requirement for RAG-dependent recombination in thymic lymphomagenesis. In contrast to expectation, the data presented here indicate that development of malignant thymic lymphoma in Atm(-/-) mice is not prevented by loss of RAG-2 and thus is not dependent on V(D)J recombination. Malignant thymic lymphomas in Rag2(-/-)Atm(-/-) mice occurred at a lower frequency and with a longer latency as compared with Atm(-/-) mice. Importantly, cytogenetic analysis of these tumors indicated that multiple chromosomal abnormalities occurred in each tumor, but that none of these involved the Tcr alpha/delta locus. Nonmalignant peripheral T cells from TCR-transgenic Rag2(-/-)Atm(-/-) mice also revealed a substantial increase in translocation frequency, suggesting that these translocations are early events in the process of tumorigenesis. These data are consistent with the hypothesis that the major mechanism of tumorigenesis in Atm(-/-) mice is via chromosomal translocations and other abnormalities that are secondary to aberrant responses to double-stranded DNA breaks. Furthermore, these data suggest that V(D)J recombination is a critical, but not essential, event during which Atm-deficient thymocytes are susceptible to developing chromosome aberrations that predispose to malignant transformation.

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.

A recurring pattern of chromosomal aberrations in mammary gland tumors of MMTV-cmyc transgenic mice.

Mice carrying the MMTV-cmyc transgene develop mammary tumors at 9 to 12 months of age. Little is known about karyotypic changes in this model of human breast cancer. We have developed and applied molecular cytogenetic techniques to study chromosomal aberrations that occur in these tumors, namely, comparative genomic hybridization and spectral karyotyping. Cell lines from eight tumors were established and analyzed, four of which carried a heterozygous p53 mutation. All of the tumor cell lines revealed increases in ploidy and/or multiple numerical and structural chromosomal aberrations. No consistent differences were observed between cmyc/p53+/+ and cmyc/p53+/- tumors, suggesting that cmyc induces karyotype instability independent of p53 status. Loss of whole chromosome (Chr) 4 was detected in five of the eight tumors. Parts of Chr 4 are syntenic to human 1p31-p36, a region that is also deleted in human breast carcinomas. Four tumors carried translocations involving the distal portion of Chr 11 (syntenic to human chromosome arm 17q), including two translocations T(X;11), with cytogenetically identical breakpoints. We compare the pattern of chromosomal aberrations with human breast cancers, find similarities in several syntenic regions, and discuss the potential of an interspecies cytogenetic map of chromosomal gains and losses.

Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation.

Cre-mediated excision of exon 11 of the breast-tumour suppressor gene Brca1 in mouse mammary epithelial cells causes increased apoptosis and abnormal ductal development. Mammary tumour formation occurs after long latency and is associated with genetic instability characterized by aneuploidy, chromosomal rearrangements or alteration of Trp53 (encoding p53) transcription. To directly test the role of p53 in Brca1-associated tumorigenesis, we introduced a Trp53-null allele into mice with mammary epithelium-specific inactivation of Brca1. The loss of p53 accelerated the formation of mammary tumours in these females. Our results demonstrate that disruption of Brca1 causes genetic instability and triggers further alterations, including the inactivation of p53, that lead to tumour formation.

Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells.

Germline mutations of the Brca1 tumor suppressor gene predispose women to breast and ovarian cancers. To study mechanisms underlying BRCA1-related tumorigenesis, we derived mouse embryonic fibroblast cells carrying a targeted deletion of exon 11 of the Brca1 gene. We show that the mutant cells maintain an intact G1-S cell cycle checkpoint and proliferate poorly. However, a defective G2-M checkpoint in these cells is accompanied by extensive chromosomal abnormalities. Mutant fibroblasts contain multiple, functional centrosomes, which lead to unequal chromosome segregation, abnormal nuclear division, and aneuploidy. These data uncover an essential role of BRCA1 in maintaining genetic stability through the regulation of centrosome duplication and the G2-M checkpoint and provide a molecular basis for the role of BRCA1 in tumorigenesis.

Adjuvant dose-intense chemotherapy with peripheral blood stem cell support in stage II-III breast cancer with five to nine involved axillary lymph nodes.

The purpose of this study is to determine outcomes for patients with high-risk nonmetastatic breast cancer undergoing high-dose chemotherapy with peripheral blood stem cell support. Forty-three patients with stage II-III disease, five to nine positive axillary lymph nodes, and a median age of 44 years (range, 27-60 years) were enrolled in a study that included: 1) standard dose doxorubicin, 5-fluorouracil, and methotrexate adjuvant therapy; 2) cyclophosphamide, etoposide, filgrastim, and peripheral blood stem cell harvest; and 3) high-dose cyclophosphamide, thiotepa, and carboplatin (CTCb) followed by peripheral blood stem cell infusion. All 43 patients received doxorubicin, 5-fluorouracil, and methotrexate, 42 (98%) received etoposide, and 41 (95%) received CTCb. Thirty-two patients (74%) are alive, 28 (65%) without relapse at a median of 55 months (range, 41-87 months). Two died (5%) of treatment-related causes, (subclavian catheter complication after etoposide and late radiation pneumonitis), and nine other deaths (21%) were associated with recurrent breast cancer. The probabilities of overall and event-free survival at 4 years were 0.77 and 0.67, respectively, compared with 0.82 and 0.69, respectively, for 72 similar patients with 10 or more positive axillary nodes receiving the same sequence of therapy. Thus, patients with five to nine positive axillary lymph nodes have a similar risk of failure after high-dose chemotherapy and peripheral blood stem cell support as patients with 10 or more positive axillary lymph nodes.

Myc/p53 interactions in transgenic mouse mammary development, tumorigenesis and chromosomal instability.

We have examined defects in mammary development and tumorigenesis in a transgenic model expressing the c-myc gene under the MMTV-LTR promoter. The stochastic tumors which arise from hyperplastic ductal and lobular lesions in this model are characterized by high rates both of apoptosis and of chromosomal instability. Since the p53 gene product is thought to be central in the maintenance of genomic integrity, in part due to its ability to induce apoptosis in cells harboring DNA damage, we examined its expression and possible mutation. Initially, we observed that unmutated p53 is strongly expressed in premalignant mammary glands and in mammary tumors derived from the MMTV-c-myc strain. We then mated the MMTV-myc strain to a p53-deficient strain as a means of examining the effect of this lesion on mammary development and tumorigenesis in the context of c-myc overexpression. A lack of both p53 alleles in the presence of c-myc overexpression resulted in a dramatic hyerplastic alteration in mammary gland development. Specifically, in female bitransgenic MMTV-c-myc/p53 null mice (MMTV-myc/p53(-/-)), lobular hyperplasias were observed at almost every ductal end bud as early as 32 days of age. In contrast, only mild ductal and lobular hyperplasias were seen in MMTV-myc mice that contained both p53 alleles (MMTV-myc/p53(+/+)); an intermediate phenotype occurred in mice with a single intact (MMTV-myc/p53(+/-)) p53 allele. Mammary carcinomas arose with a high frequency in MMTV-myc/p53(+/-) mice; the tumors were comparable in frequency, histology and apoptotic index to the tumors in MMTV-myc/p53(+/+) mice. Also, as previously observed (Elson et al., 1995), lymphomas arose with extremely short latency in MMTV-myc/p53(-/-) mice, precluding study of the fate of their hyperplastic mammary lesions in situ. The frequency of p53 mutations in MMTV-myc/p53(+/+) and MMTV-myc/p53(+/-) mammary tumors and in cell lines derived from these tumors was examined by direct sequencing. No point mutations or deletions in p53 were observed in mammary tumors or cell lines from either genotype. Finally, a detailed chromosomal analysis using multicolor spectral karyotyping (SKY) revealed that there were multiple chromosomal alterations in the c-myc-overexpressing cells that contained either one or two unmutated p53 alleles. Variable ploidy changes, a common translocation of chromosome 11, and other chromosomal aberrations were observed. Our data thus support an interaction between c-Myc and p53 in mammary development, but suggest that loss of p53 is required neither for c-myc-dependent tumorigenesis nor for c-myc-dependent chromosomal instability.

Second attempts at mobilization of peripheral blood stem cells in patients with initial low CD34+ cell yields.

The purpose of this study was to determine the effectiveness of second mobilization strategies in patients who yielded < 2.5 x 10(6) CD34+ PBSC/kg after initial mobilization. Repeat mobilization attempts were made with chemotherapy and G-CSF (n = 61) or G-CSF alone (n = 58) in patients who failed initial mobilization with chemotherapy and G-CSF (n = 92) or G-CSF alone (n = 27). A median of 0.27 x 10(6) CD34+ cells/kg per apheresis was collected after the second mobilization, compared with 0.16 with initial harvests (p = 0.0001). Forty-eight percent achieved a target CD34+ cell dose > or = 2.5 x 10(6)/kg when harvests from the first and second mobilizations were combined. Fifteen of 17 patients (88%) with > or = 1.5 x 10(6) CD34+ cells/kg harvested after first mobilization had > or = 2.5 x 10(6) CD34+ cells/kg collected when first and second harvests were combined, as compared with 42 of 102 (41%) achieving < 1.5 x 10(6) CD34+ cells/kg with first PBSC harvests (p = 0.0001). Second mobilizations with chemotherapy and G-CSF or G-CSF alone resulted in similar CD34+ cell yields. Toxicities of second mobilizations were comparable with those of first mobilizations. Seventy-nine patients (66%) received high-dose chemotherapy with PBSC support, with recovery of neutrophils and platelets in a median of 11 and 15 days, respectively. Transplant-related mortality was 4%, and event-free survival at 2 years was 0.34. It was concluded that second mobilization attempts in patients who fail to achieve > or = 2.5 x 10(6) CD34+ cells/kg on initial mobilization were successful in 48% of patients. G-CSF alone was as effective as chemotherapy plus G-CSF in mobilizing CD34+ cells and was associated with less morbidity.

A targeted disruption of the murine Brca1 gene causes gamma-irradiation hypersensitivity and genetic instability.

Germline mutations of the Brcal gene are responsible for most cases of familial breast and ovarian cancers, but somatic mutations are rarely detected in sporadic events. Moreover, mouse embryos deficient for Brca1 have been shown to die during early embryogenesis due to a proliferation defect. These findings seem incompatible with the tumor suppress function assigned to this gene and raise questions about the mechanism by which Brca1 mutations cause tumorigenesis. We now directly demonstrate that BRCA1 is responsible for the integrity of the genome. Murine embryos carrying a Brca1 null mutation are developmentally retarded and hypersensitive to gamma-irradiation, suggesting a failure in DNA damage repair. This notion is supported by spectral karyotyping (SKY) of metaphase chromosomes, which display numerical and structural aberrations. However, massive chromosomal abnormalities are only observed when a p53-/- background is introduced. Thus, a p53 dependent cell cycle checkpoint arrests the mutant embryos and prevents the accumulation of damaged DNA. Brca1-/- fibroblasts are not viable, nor are Brca1-/-:p53-/- fibroblasts. However, proliferative foci arise from Brca1-/-: p53-/- cells, probably due to additional mutations that are a consequence of the accumulating DNA damage. We believe that the increased incidence of such additional mutations accounts for the mechanism of tumorigenesis associated with Brca1 mutations in humans.

Previously hidden chromosome aberrations in T(12;15)-positive BALB/c plasmacytomas uncovered by multicolor spectral karyotyping.

The majority of BALB/c mouse plasmacytomas harbor a balanced T(12;15) chromosomal translocation deregulating the expression of the proto-oncogene c-myc. Recent evidence suggests that the T(12;15) is an initiating tumorigenic mutation that occurs in early plasmacytoma precursor cells. However, the possible contribution of additional chromosomal aberrations to the progression of plasmacytoma development has been largely ignored. Here we use multicolor spectral karyotyping (SKY) to evaluate 10 established BALB/c plasmacytomas in which the T(12;15) had been previously detected by G banding. SKY readily confirmed the presence of this translocation in all of these tumors and in three plasmacytomas newly identified secondary cytogenetic changes of the c-myc-deregulating chromosome (Chr) T(12;15). In addition, numerous previously unknown aberrations were found to be scattered throughout the genome, which was interpreted to reflect the general genomic instability of plasmacytomas. Instability of this sort was not uniform, however, because only half of the tumors were heavily rearranged. Seven apparent hot spots of chromosomal rearrangements (40% incidence) were identified and mapped to Chrs 1B, 1G-H, 2G-H1, 4C7-D2, 12D, 14C-D2, and XE-F1. Two of these regions, Chr 1B and Chr 4C7-D2, are suspected to harbor plasmacytoma susceptibility loci; Pctr1 and Pctr2 on Chr 4C7-D2 and as yet unnamed loci on Chr 1B. These results suggest that secondary chromosomal rearrangements contribute to plasmacytoma progression in BALB/c mice. To evaluate the biological significance of these rearrangements, SKY will be used in follow-up experiments to search for the presence of recurrent and/or consistent secondary cytogenetic aberrations in primary BALB/c plasmacytomas.

Genomic characterization of a testis-specific TFIIS (TCEA2) gene.

There is a family of genes encoding TFIIS-related proteins in human cells. We have focused upon the genomic organization of one family member expressed primarily in the testis. This gene encodes a transcription elongation factor similar to but distinct from that encoded by a previously reported TFIIS gene. Also in contrast to the previously reported TFIIS gene, the testis gene contains introns. All exon-intron junction sequences match the consensus GT/AG rule. The gene consists of seven exons and six introns with a total size of approximately 7 kb. The nucleotide sequence of the 5 flanking region of the testis TFIIS gene contains several potential regulatory factor-binding sites, not all of which are present in the TFIIS gene, whose expression is nearly ubiquitous. Elucidation of the full structure of the testis TFIIS gene should be useful for determining its chromosomal localization and its potential role in the regulation of gene expression in human tissues.