Heterogeneous mass distribution of the rubble-pile asteroid (101955) Bennu.

The gravity field of a small body provides insight into its internal mass distribution. We used two approaches to measure the gravity field of the rubble-pile asteroid (101955) Bennu: (i) tracking and modeling the spacecraft in orbit about the asteroid and (ii) tracking and modeling pebble-sized particles naturally ejected from Bennu's surface into sustained orbits. These approaches yield statistically consistent results up to degree and order 3, with the particle-based field being statistically significant up to degree and order 9. Comparisons with a constant-density shape model show that Bennu has a heterogeneous mass distribution. These deviations can be modeled with lower densities at Bennu's equatorial bulge and center. The lower-density equator is consistent with recent migration and redistribution of material. The lower-density center is consistent with a past period of rapid rotation, either from a previous Yarkovsky-O'Keefe-Radzievskii-Paddack cycle or arising during Bennu's accretion following the disruption of its parent body.

A partially differentiated interior for (1) Ceres deduced from its gravity field and shape.

Remote observations of the asteroid (1) Ceres from ground- and space-based telescopes have provided its approximate density and shape, leading to a range of models for the interior of Ceres, from homogeneous to fully differentiated. A previously missing parameter that can place a strong constraint on the interior of Ceres is its moment of inertia, which requires the measurement of its gravitational variation together with either precession rate or a validated assumption of hydrostatic equilibrium. However, Earth-based remote observations cannot measure gravity variations and the magnitude of the precession rate is too small to be detected. Here we report gravity and shape measurements of Ceres obtained from the Dawn spacecraft, showing that it is in hydrostatic equilibrium with its inferred normalized mean moment of inertia of 0.37. These data show that Ceres is a partially differentiated body, with a rocky core overlaid by a volatile-rich shell, as predicted in some studies. Furthermore, we show that the gravity signal is strongly suppressed compared to that predicted by the topographic variation. This indicates that Ceres is isostatically compensated, such that topographic highs are supported by displacement of a denser interior. In contrast to the asteroid (4) Vesta, this strong compensation points to the presence of a lower-viscosity layer at depth, probably reflecting a thermal rather than compositional gradient. To further investigate the interior structure, we assume a two-layer model for the interior of Ceres with a core density of 2,460-2,900 kilograms per cubic metre (that is, composed of CI and CM chondrites), which yields an outer-shell thickness of 70-190 kilometres. The density of this outer shell is 1,680-1,950 kilograms per cubic metre, indicating a mixture of volatiles and denser materials such as silicates and salts. Although the gravity and shape data confirm that the interior of Ceres evolved thermally, its partially differentiated interior indicates an evolution more complex than has been envisioned for mid-sized (less than 1,000 kilometres across) ice-rich rocky bodies.

Cleavage of the MLL gene by activators of apoptosis is independent of topoisomerase II activity.

Exposure to topoisomerase II inhibitors is linked to the generation of leukemia involving translocations of the MLL gene, normally restricted to an 8.3 kbp tract, the breakpoint cluster region (BCR). Using an in vitro assay, apoptotic activators, including radiation and anti-CD95 antibody, trigger site-specific cleavage adjacent to exon 12 within the MLL BCR and promote translocation of the MLL gene in cells that can survive. To explore the mechanism of cleavage and rearrangement in more detail, the entire MLL BCR was placed into the pREP4 episomal vector and transfected into human lymphoblastoid TK6 cells. Episomes containing either the MLL BCR, or deletion constructs of 367 bp or larger, were cleaved at the same position as genomic MLL after exposure to apoptotic stimuli. Further analysis of sequence motifs surrounding the cleaved region of MLL showed the presence of both a predicted nuclear matrix attachment sequence and a potential strong binding site for topoisomerase II, flanking the site of cleavage. Inactivation of topoisomerase II by the catalytic inhibitor merbarone did not inhibit MLL cleavage, suggesting that the initial cleavage step for MLL rearrangement is not mediated by topoisomerase II.

Surviving apoptosis.

The concept that cells subjected to chromatin cleavage during apoptosis are destined to die is being challenged. The execution phase of apoptosis is characterized by the activation of effector caspases, such as caspase-3, that cleave key regulatory or structural proteins and in particular activate apoptotic nucleases such as the caspase activated deoxyribonuclease (CAD). It is apparent that caspases of this type may become active both through non-apoptotic processing and potentially within cells that exhibit apoptotic morphology but are subsequently able to survive. In such systems caspase suppressor molecules, the inhibitors of apoptotic proteins or IAP's, may rescue cells from apoptotic nuclease(s) attack initiated by transient caspase activation. The MLL gene is involved in leukemogenic translocations in ALL and AML and is a target of nuclease cleavage during apoptosis. Translocations initiated at the site of apoptotic nuclease attack within MLL have been identified and may offer a model, with clinical relevance, for DNA damage mediated by the apoptosis system in cells destined to survive. The specificity of apoptotic cleavage combined with the potential for recovery from the execution phase of apoptosis suggests a novel and pathogenic role for apoptosis in creating translocations with leukemogenic potential.

Steroid-mediated inhibition of radiation-induced apoptosis in C4-1 cervical carcinoma cells is p53-dependent.

In human papillomavirus (HPV) infected cervical epithelial cells the synthetic steroid dexamethasone inhibits radiation-induced apoptosis and increases the transcription of HPV E6/E7, enhancing p53 degradation. The aim of this study was to determine if suppression of apoptosis was mechanistically linked to changes in p53. HPV 16 E6 or E6/E7 expression vectors were transiently transfected into C4-1 HPV 18-positive cervical carcinoma cells to mimic the enhanced transcription following steroid treatment. After irradiation, apoptosis was suppressed in these cells comparable to the effect observed after steroid treatment alone. To confirm whether loss of p53 was responsible for the inhibition of apoptosis, residual p53 in C4-1 cells was targeted by stable transfection with a dominant-negative p53 mutant. While radiation-induced apoptosis increased after mutant transfection, inhibition of programmed cell death by steroid treatment was either eliminated or substantially reduced. Steroid-dependent inhibition of radiation-induced apoptosis in carcinoma of the cervix involves E6 modulation of p53 expression and may adversely affect treatment.

Apoptotic triggers initiate translocations within the MLL gene involving the nonhomologous end joining repair system.

Translocations involving the MLL gene at 11q23 are a frequent finding in therapy-related leukemia and are concentrated within a short, 8.3-kb tract of DNA, the breakpoint cluster region. In addition, a specific site adjacent to exon 12 within this region of MLL is cleaved in cells undergoing apoptosis. We show here, using human TK6 lymphoblastoid cells, that irradiation and the apoptotic trigger anti-CD95 antibody are each able to initiate translocations at the MLL exon 12 cleavage site. The translocation junctions produced contain regions of microhomology consistent with operation of the nonhomologous end joining (NHEJ) repair process. Participation of the NHEJ process is supported by the identification of the NHEJ component DNA-PKcs at the site of apoptotic cleavage. Suppression of DNA-PKcs function by the phosphatidylinositol 3-kinase inhibitor wortmannin compromises DNA end joining, increases site-specific cleavage within MLL, and eliminates MLL-restricted translocations. We propose that activation of apoptotic effector nucleases alone is sufficient to generate proleukemogenic translocations and raises the possibility that some of these may persist in cells that evade apoptotic execution and survive.

LINE L1 retrotransposable element is targeted during the initial stages of apoptotic DNA fragmentation.

Using a directional cloning strategy, DNA sequence information was obtained corresponding to the site of early radiation-induced apoptotic DNA fragmentation within the human lymphoblastoid cell line TK6. Data were obtained from 88 distinct clones comprising approximately 65 kbp of sequenced material. Analysis of all cloned material showed that sequences in the 10 bp immediately adjacent to the cleavage sites were enriched in short oligoT tracts. The proportion of repetitive DNA within the entire cloned material was found to be within the normal range. However the distribution of Alu and LINE repetitive DNA were biased to positions at or adjacent to the apoptotic cleavage site. In particular, a non-random distribution of five cleavage sites was found clustered within the second ORF of the LINE L1 that partially overlapped with two binding sites for the nuclear matrix-associated protein SATB1. Three other clones, containing alpha satellite elements, were also linked to a DNA matrix binding function. These data indicate that the site of chromatin loop formation at the nuclear matrix may be a specific target for early DNA fragmentation events during apoptosis.

Inhibition of radiation-induced apoptosis by dexamethasone in cervical carcinoma cell lines depends upon increased HPV E6/E7.

Through a glucocorticoid-responsive promoter, glucocorticoids can regulate the transcription of the human papillomavirus (HPV) E6 and E7 viral genes which target the tumour suppressor proteins p53 and Rb respectively. In C4-1 cells, the glucocorticoid dexamethasone up-regulated HPV E6/E7 mRNA and decreased radiation-induced apoptosis. In contrast, dexamethasone had no effect on apoptosis of cells that either lack the HPV genome (C33-a) or in which HPV E6/E7 transcription is repressed by dexamethasone (SW756). Irradiated C4-1 cells showed increased p53 expression, while dexamethasone treatment prior to irradiation decreased p53 protein expression. In addition, p21 mRNA was regulated by irradiation and dexamethasone in accordance with the observed changes in p53. Overall, glucocorticoids decreased radiation-induced apoptosis in cervical carcinoma cells which exhibit increased HPV E6/E7 transcription and decreased p53 expression. Therefore, in HPV-infected cervical epithelial cells, p53-dependent apoptosis appears to depend upon the levels of HPV E6/E7 mRNA.

RU486 increases radiosensitivity and restores apoptosis through modulation of HPV E6/E7 in dexamethasone-treated cervical carcinoma cells.

OBJECTIVE: Cervical carcinoma tumors containing radioresistant cells are associated with decreased local control and survival. Therefore, strategies to increase cell kill during radiotherapy have a clear rationale. It was previously determined that treatment with the corticosteroid dexamethasone increased radioresistance and decreased apoptosis in C4-1 cervical carcinoma cells. The goal of this study was to determine whether hormone antagonists, specifically Mifepristone (RU486), could reverse the effects of dexamethasone on clonogenic survival and apoptosis following gamma-irradiation. METHODS: Cervical carcinoma cell line C4-1 cells were exposed to 1 microM dexamethasone in the presence or absence of 1 microM Mifepristone (RU486), a hormone antagonist, and irradiation. Cells were analyzed for steroid-dependent HPV E6/E7 mRNA expression (by Northern blot analysis), clonogenic survival, and apoptosis (by Annexin V staining and the DNA fragmentation assay). In addition, p53 protein levels were determined by Western blot analysis. RESULTS: The hormone antagonist RU486 reversed dexamethasone-dependent upregulation of E6/E7 mRNA and restored radiation-induced p53 expression, apoptosis, and clonogenic survival to levels similar to those observed following irradiation alone. CONCLUSION: RU486 reverses glucocorticoid-dependent upregulation of HPV E6/E7, which corresponds to restoration of p53 expression, and restores radiosensitivity and apoptosis following gamma-irradiation. Therefore, it appears that along with radiation, RU486 may be a beneficial agent in the treatment of hormone-reactive cervical tumors.

P53 overexpression is associated with bulky tumor and poor local control in T1 glottic cancer.

PURPOSE: To study the role of two possible prognostic factors, p53 and tumor bulk, and their interaction with other tumor and treatment variables in early-stage laryngeal cancer patients treated with curative radiotherapy. METHODS: One hundred two patients with T1N0M0 squamous cell carcinoma of the glottic larynx treated with definitive radiotherapy were analyzed. p53 status in pretreatment biopsy specimens was assessed by immunohistochemistry (IHC) using mouse monoclonal antibody DO-7. Tumors were classified as small surface lesions or bulky tumors. All tumor-related and treatment-related variables which might influence the outcome were analyzed. Local control after definitive radiotherapy was the end point of the study. RESULTS: The local control at 5 years for the entire group of patients was 78% (80/102) and 91% (93/102) after surgical salvage. p53 overexpression by IHC was seen in 37% (38/102) of patients. Tumors were classified as small volume in 69 (68%) and bulky in 33 (32%) patients. Five-year local control was 48% for p53-positive patients as compared to 94% for p53-negative patients (p = 0.0001). Tumor bulk was the other important prognostic factor, with 5-year local control of 91% for small tumors and 48% for bulky tumors (p = 0.0001). Patients who had both p53 positivity and bulky tumors did worse, with a 5-year local control of 23% as compared to 92% for all other groups combined (p = 0.0001). Among other variables, only the length of radiation time was of borderline significance. CONCLUSION: Both p53 overexpression and tumor bulk are independent prognostic factors for local control in early-stage glottic cancer treated with curative radiotherapy. The precise relationship between a genetic event, the p53 mutation, and an observable phenotype expression such as tumor bulk needs to be further defined.

Abortive apoptosis as an initiator of chromosomal translocations.

Apoptosis is a well-recognized regulator of a cell populations size and structure. Irreversible stages of apoptosis lead to activation of different enzymatic cascades, changes in cell morphology and DNA fragmentation. However, little is known about nuclear events which accompany the initial stages of apoptosis. These events are connected with introduction of limited amounts of double strand breaks into genomic DNA, some of which may be subsequently rejoined. We hypothesize here that the initial stages of apoptotic DNA fragmentation may be reversible and connected with the initiation of recombinational events and certain chromosomal translocations. The factors influencing apoptosis reversibility and cell survival after delivery of apoptotic stimuli may provide new insights into mechanisms of lymphocyte development and tumorigenesis.

Second primary tumors in laryngeal cancer: results of long-term follow-up.

PURPOSE: The development of second primary tumors (SPTs) is the most important factor determining the survival in early-stage head and neck cancer patients, whose first tumor has been successfully treated. New methods of examining genetic changes have raised doubts about the validity of the widely held field cancerization hypothesis as the cause of SPTs, and an alternative hypothesis of monoclonal origin has been proposed. The objectives of this study were to look at the pattern of development of SPTs and the possible factors influencing the incidence of SPTs and the survival in early-stage laryngeal cancer with long-term follow-up. METHODS AND MATERIALS: One hundred forty-four consecutive patients of T1N0M0 squamous cell carcinoma of the true vocal cord treated with definitive radiotherapy between 1976 and 1992 were analyzed. The incidence, time to development, and survival of aerodigestive and other SPTs were noted. p53 overexpression indicating a mutated p53 gene was analyzed by immunohistochemistry. RESULTS: With a median follow-up of 6 years (range 2-20 years), 42 patients developed a SPT, 24 in upper aerodigestive tract and lung and 18 at other sites. The actuarial incidence of developing a SPT at 5, 10, and 15 years was 23%, 44%, and 48.7% respectively. The median time interval for development of SPT in an upper aerodigestive tract was 21 months as opposed to 50 months for other sites (p = 0.02). The most common sites of SPTs included lung for upper aerodigestive tract; and prostate, followed by colon, for other sites. The actuarial risk of developing a nonaerodigestive SPT at 5 and 10 years was 35% and 55% respectively. p53 status affected neither the incidence of SPT nor the survival. SPTs were the leading cause of death in these early-stage laryngeal cancer patients. CONCLUSION: The origin of SPTs seems to be multifactorial, involving both the field cancerization effect and an increased baseline genetic predisposition. Until more reliable genetic markers are developed, chemoprevention remains the best treatment option at preventing SPTs in these early-stage patients.

Association between DNA cleavage during apoptosis and regions of chromatin replication.

We have addressed the association between the site of DNA cleavage during apoptosis and DNA replication. DNA double strand breaks were introduced into chromatin containing pulse labeled nascent DNA by the induction of apoptosis or autocleavage of isolated nuclei. The location of these breaks in relation to nascent DNA were revealed by Bal31 exonuclease digestion at the cut sites. Our data show that Bal31 accessible cut sites are directly linked to regions enriched in nascent DNA. We suggest that these regions coincide with the termini of replication domains, possibly linked by strong DNA-matrix interactions with biophysically defined topological structures of 0.5-1.3 Mbp in size. The 50 kbp fragments that are commonly observed as products of apoptosis are also enriched in nascent DNA within internal regions but not at their termini. It is proposed that these fragments contain a subset of replicon DNA that is excised during apoptosis through recognition of their weak attachment to the nuclear matrix within the replication domain.

Synchronous block in DNA synthesis initiation with change in chromatin topology mediated by VP16.

The impact of chromatin topology on the DNA synthetic process was studied in the human squamous-cell carcinoma cell line SQ-20B. A 1-h exposure < or = 10 microM VP16 produced an increase in DNA supercoil tension, measured by recording laser light scatter from salt-extracted nuclei. This change was precisely paralleled by a decrease in DNA synthesis. The effects on both DNA supercoiling and DNA synthesis were suppressed at VP16 concentrations between 10 and 20 microM. The changes in DNA supercoiling and synthesis at VP16 concentrations -10 microM were eliminated by coincubation with mimosine, a DNA synthesis initiator poison. We conclude that brief exposure to low concentrations of VP16 disturbs the balance of torsional energy within discrete replicon domains by affecting normal topoisomerase II activity at sites of replication initiation. The resultant increase in negative supercoil tension mediates a topologic checkpoint, limiting the initiation of DNA synthesis. Such a checkpoint may be a common pathway for control, both during the normal replicative cycle and subsequent to DNA damage.

Mechanisms of induction of apoptotic DNA fragmentation.

PURPOSE: Despite its common use as an indicator of apoptosis, little is known about the mechanisms controlling apoptotic DNA fragmentation in irradiated cells. This review discusses the pathways of chromatin fragmentation, and the role of both nucleases and chromatin structure in this process. DEFINITIONS: DNA fragmentation linked to apoptosis is a combination of cleavage events excising both large DNA fragments within the range 0.4-1.0 Mbp and 50 kbp followed by random cuts within internucleosomal regions (i.e. DNA laddering). The first two cleavage steps can be detected in virtually all apoptotic cells, but DNA laddering is not ubiquitously observed. Endonucleases that mediate this cleavage of chromatin may be classified by substrate specificity, mode of DNA cleavage and their cofactor requirements. CONCLUSIONS: Three major pathways of DNA fragmentation are proposed and discussed: (1) upregulation of endonucleases, (2) their intranuclear/intracellular redistribution and (3) primary changes of chromatin structure.

Transfection of a vector expressing wild-type p53 into cells of two human glioma cell lines enhances radiation toxicity.

Replication-deficient adenovirus (Adv5)-based vectors containing either wild-type p53 or the beta-gal marker gene were introduced into cells of the T98G (p53 mutant) and U87MG (p53 wild-type) human glioma cell lines. The wild-type p53 gene was successfully expressed in each cell line as shown by flow cytometry and Western blotting. The presence of the p53-expressing vector was toxic in both cell lines compared to control cells or to those containing the beta-gal vector. At levels of Adv5p53 vector that produced detectable toxicity, the effect of irradiation was enhanced, producing a twofold increase in cell killing. In the T98G cells, the presence of the p53 vector resulted in an increase in the number of cells undergoing apoptosis after irradiation, whereas a smaller and only additive response was observed in the U87MG cells. Conversely, an increase in micronucleus formation, indicating corrupt mitotic activity, was observed in irradiated Adv5p53-positive U87MG cells but not in T98G cells. These data suggest that p53-expressing vectors effectively enhance radiation lethality in these human glioma cell lines, but that the mechanism of action cannot be simply related to activation of the p53-dependent pathway to apoptosis.

Multiple forms of endonuclease activity linked with radiation induced apoptosis in C4-1 cervical carcinoma cells.

Irradiation of C4-1 cervical carcinoma cells induced apoptosis, as determined by their morphology and the presence of oligonucleosomal DNA fragmentation, with the formation of 5'- P and 3'-OH termini. Extracts of nuclear proteins from both control and irradiated cells possessed similar metallodependent endonucleolytic activity which cleaved target plasmid DNA with the same specificity as that found in apoptotic cells. Fractionation of the nuclear extracts revealed that the predominant endonuclease activity of unirradiated cells was a protein of approximately 40 kDa. After irradiation, the predominant activity was found to be associated with a 70 kDa fraction, with a reduction in the 40 kDa form. The activity of each endonuclease was found to be Ca2+ and Mg2+ dependent. It is proposed that the changes in molecular weight observed for these enzymes may be linked to the final step in apoptosis execution, irreversible chromatin fragmentation, and thus offer a potentially novel target for manipulating the effector pathway of apoptosis in these cells.

Is p53 an independent prognostic factor in patients with laryngeal carcinoma?

BACKGROUND: Carcinoma of the true vocal cord represents the earliest clinically recognizable invasive malignancy in the head and neck region and provides a unique model for studying possible prognostic genetic markers. The aim of this study was to determine whether p53 overexpression correlated with tumor recurrence in a homogenous population of patients with early stage glottic carcinoma treated with radiotherapy alone. METHODS: One hundred and fourteen patients with T1N0M0 squamous cell carcinoma of the glottis were treated with curative radiotherapy between 1976 and 1990. With a median follow-up of 6 years, actuarial local control was 80% with 23 local recurrences. Laryngeal biopsy specimens obtained prior to radiation therapy were analyzed retrospectively in 22 patients. Forty-five patients with local control were used as a control group. p53 overexpression indicating a mutated p53 gene was analyzed by immunohistochemistry using the mouse monoclonal antibody D0-7. RESULTS: Approximately 82% of carcinomas that recurred locally expressed p53 compared with only 29% of those with local control (P < 0.001). No significant relation was noted between p53 expression and histologic grade. Intensity of staining did not predict tumor recurrence. CONCLUSIONS: The authors believe that this case-controlled study demonstrated the role of p53 as an independent prognostic factor in patients with early stage glottic carcinoma.

Mycoplasma infection can sensitize host cells to apoptosis through contribution of apoptotic-like endonuclease(s).

Mycoplasma infection may lead to various pathologies in a broad range of hosts. It has been shown that Mycoplasma may trigger cell death in cell cultures; however, the mechanism remains unknown. In the present paper we show that Mycoplasma infection of different lymphocyte and epithelial tumour cell lines leads to the inhibition of proliferation, and increased cell death, accompanied by DNA fragmentation and the morphological features of apoptosis. We also showed that this infection leads to an increased sensitivity of cells to various inducers of apoptosis targeting different signalling pathways. Finally, we show that increased apoptosis is associated with overexpression of an endonuclease produced by Mycoplasma. This endonuclease is recovered in the nuclear fraction of host cells, introduces mostly DSB and is active at neutral pH in the presence of divalent cations. Activation of this endonuclease is connected with limited proteolysis, which may be reproduced in vitro by snake venom serine proteinase.