New factors in mammalian DNA repair-the chromatin connection.

In response to DNA damage mammalian cells activate a complex network of stress response pathways collectively termed DNA damage response (DDR). DDR involves a temporary arrest of the cell cycle to allow for the repair of the damage. DDR also attenuates gene expression by silencing global transcription and translation. Main function of DDR is, however, to prevent the fixation of debilitating changes to DNA by activation of various DNA repair pathways. Proper execution of DDR requires careful coordination between these interdependent cellular responses. Deregulation of some aspects of DDR orchestration is potentially pathological and could lead to various undesired outcomes such as DNA translocations, cellular transformation or acute cell death. It is thus critical to understand the regulation of DDR in cells especially in the light of a strong linkage between the DDR impairment and the occurrence of common human diseases such as cancer. In this review we focus on recent advances in understanding of mammalian DNA repair regulation and a on the function of PAXX/c9orf142 and ZNF281 proteins that recently had been discovered to play a role in that process. We focus on regulation of double-strand DNA break (DSB) repair via the non-homologous end joining pathway, as unrepaired DSBs are the primary cause of pathological cellular states after DNA damage. Interestingly these new factors operate at the level of chromatin, which reinforces a notion of a central role of chromatin structure in the regulation of cellular DDR regulation.

Silencing of endogenous IGFBP-5 by micro RNA interference affects proliferation, apoptosis and differentiation of neuroblastoma cells.

Signal transduction through the IGF axis is implicated in proliferation, differentiation and survival during development and adult life. The IGF axis includes the IGF binding proteins (IGFBPs) that bind IGFs with high affinity and modulate their activity. In neuroblastoma (NB), a malignant childhood tumor, we found that IGFBP-5 is frequently expressed. Since NB is an IGF2-sensitive tumor, we investigated the relevance and the function of endogenous IGFBP-5 in LAN-5 and in SY5Y(N) cell lines transfected with micro and small interfering RNAs directed to IGFBP-5 mRNA. Cells in which IGFBP-5 expression was suppressed were growth-inhibited and more prone to apoptosis than the parental cell line and controls. Apoptosis was further enhanced by X-ray irradiation. The ability of these cells to undergo neuronal differentiation was impaired after IGFBP-5 inhibition but the effect was reversed by exposure to recombinant IGFBP-5. Together, these data demonstrate the importance of IGFBP-5 for NB cell functions and suggest that IGFBP-5 might serve as a novel therapeutic target in NB.

DR-nm23 gene expression in neuroblastoma cells: relationship to integrin expression, adhesion characteristics, and differentiation.

BACKGROUND: Neuroblastoma, a childhood tumor originating from cells of the embryonic neural crest, retains the ability to differentiate, yielding cells with epithelial-Schwann-like, neuronal, or melanocytic characteristics. Since nm23 gene family members have been proposed to play a role in cellular differentiation, as well as in metastasis suppression, we investigated whether and how DR-nm23, a recently identified third member of the human nm23 gene family, might be involved in neuroblastoma differentiation. METHODS: Three neuroblastoma cell lines (human LAN-5, human SK-N-SH, and murine N1E-115) were used in these experiments; cells from two of the lines (SK-N-SH and N1E-115) were also studied after being stably transfected with a plasmid containing a full-length DR-nm23 complementary DNA. Cellular expression of specific messenger RNAs and proteins was assessed by use of standard techniques. Cellular adhesion to a variety of protein substrates was also evaluated. RESULTS: DR-nm23 messenger RNA levels in nontransfected LAN-5 and SK-N-SH cells generally increased with time after exposure to differentiation-inducing conditions; levels of the other two human nm23 messenger RNAs (nm23-H1 and nm23-H2) remained essentially constant. Transfected SK-N-SH cells overexpressing DR-nm23 exhibited some characteristics of differentiated cells (increased vimentin and collagen type IV expression) even in the absence of differentiation-inducing conditions. Compared with control cells, DR-nm23-transfected cells exposed to differentiation-inducing conditions showed a greater degree of growth arrest (SK-N-SH cells) and greater increases in integrin protein expression, especially of integrin beta1 (N1E-115 cells). DR-nm23-transfected N1E-115 cells also showed a marked increase in adhesion to collagen type I-coated tissue culture plates that was inhibited by preincubation with an anti-integrin beta1 antibody. CONCLUSIONS: DR-nm23 gene expression appears to be associated with differentiation in neuroblastoma cells and may affect cellular adhesion through regulation of integrin protein expression.

Coexpression of messenger RNA for TRK protooncogene and low affinity nerve growth factor receptor in neuroblastoma with favorable prognosis.

Nerve growth factor (NGF), essential for differentiation and survival of sympathetic neurons is suggested to play a role in differentiation or regression of neuroblastoma. Expression of mRNA for the trk protooncogene, encoding a tyrosine kinase receptor essential for functional NGF signal transduction, and mRNA for the low affinity NGF receptor (LNGFR) was examined in 45 neuroblastomas and 3 benign ganglioneuromas using Northern blot analysis. Expression of trk mRNA and LNGFR mRNA correlated with young age, favorable clinical stages, and absence of N-myc amplification. All children (n = 19) with neuroblastomas coexpressing mRNA for trk and LNGFR are alive 8-84 months from diagnosis, regardless of age and stage. In contrast, no child (n = 15) with tumor lacking trk mRNA is alive without disease. Three subsets of patients were distinguished, one favorable (trk+, LNGFR+, n = 19, 100% survival probability), one intermediate (trk+, LNGFR-, n = 11, 62.3% survival probability), and one unfavorable (trk-, LNGFR +/-, n = 15, 0% survival probability, P < 0.001). In widespread neuroblastoma stage IVS prone to spontaneous regression, three tumors coexpressing trk and LNGFR mRNAs regressed after no or minimal therapy while the remaining tumor expressing trk but not LNGFR mRNA progressed to a fatal outcome. It is concluded that neuroblastomas coexpressing mRNA for both NGF receptor subtypes are favorable tumors likely to differentiate or regress spontaneously or respond to conventional therapy. It is further hypothesized that loss of functional NGF receptors is an important step in tumorigenesis of undifferentiated malignant childhood neuroblastoma. For these unfavorable tumors current therapy remains futile and first-line innovative therapy is justified.

Inhibition of proliferation by c-myb antisense RNA and oligodeoxynucleotides in transformed neuroectodermal cell lines.

Transfection of a neuroblastoma cell line with expression vectors containing two different segments of human c-myb complementary DNA in antisense orientation yielded far fewer transfectant clones than did the transfection with the identical segments in sense orientation. In cell clones expressing c-myb antisense RNA, levels of the c-myb protein were down-regulated and the proliferation rate was slower than that of cells transfected with sense constructs or the untransfected parental cell line. Treatment of neuroblastoma and neuroepithelioma cell lines with a c-myb antisense oligodeoxynucleotide strongly inhibited cell growth. These data indicate a definite involvement of c-myb in the proliferation of neuroectodermal tumor cells extending the role of this protooncogene beyond the hematopoietic system. The availability of cell clones that transcribe c-myb antisense RNA provides a useful tool to study the involvement of other genes in the proliferation and differentiation of neuroblastoma cells.

Expression of B-myb in neuroblastoma tumors is a poor prognostic factor independent from MYCN amplification.

The transcription factors of the Myb family are expressed in several tissues and play an important role in cell proliferation, differentiation, and survival In this study, the expression of A-myb, B-myb, and c-myb was investigated in a group of 64 neuroblastomas at different dinical stages by a sensitive reverse transcription-PCR tchnique and correlated with patients' survival. All of the myb genes were frequently expressed in neuroblastoma tumors. Interestingly, the expression of B-myb, which was detected in 33 cases, was associated with an increased risk of death (P = 0.027 in a univariate analysis), whereas there was no correlation with A-myb and c-myb expression. In addition, in a multivariate Cox regression analysis that included myb gene expression, MYCN status, age at diagnosis, and tumor staging, MYCN amplification and B-myb expression were independently associated to an increased risk (P < 0.01 and P = 0.015, respectively). In overall survival curves obtained by stratifying the neuroblastoma cases on the basis of MYCN status and B-myb expression, the group of patients without MYCN amplification and positive for B-myb expression had worse survival probability than that without MYCN amplification and nonexpressing B-myb (P < 0.01). In summary, these findings provide the first demonstration that B-myb expression can be a useful prognostic marker in human neuroblastoma. Moreover, B-myb expression has a prognostic value complementary to MYCN amplification and can identify a group of high-risk patients that would not be predicted on the basis of the MYCN status only.

Lack of correlation between N-myc and MAX expression in neuroblastoma tumors and in cell lines: implication for N-myc-MAX complex formation.

Detectable levels of MAX messenger RNA were found in a set of human neuroblastoma tumors and established cell lines. MAX mRNA levels were independent of tumor stage and N-myc genomic amplification. By contrast, N-myc mRNA transcripts were detectable only in tumors with amplification of N-myc gene and in cell lines. Analysis by reverse transcriptase polymerase chain reaction and hybridization to specific oligodeoxynucleotide probes revealed approximately equal amounts of two MAX transcripts in all cases analyzed. Immunoprecipitations with a specific antibody to MAX detected two proteins of M(r) 21,000 and 22,000 in approximately equal amounts in all neuroblastoma lines regardless of N-myc amplification and/or expression. On the other hand, protein binding to the myc DNA consensus sequence correlated with N-myc expression in neuroblastoma cells. Thus, N-myc expression might be a limiting factor in the formation of the N-myc-MAX heterodimer in neuroblastomas.

ZNF281 contributes to the DNA damage response by controlling the expression of XRCC2 and XRCC4.

ZNF281 is a zinc-finger factor involved in the control of cellular stemness and epithelial-mesenchymal transition (EMT). Here, we report that ZNF281 expression increased after genotoxic stress caused by DNA-damaging drugs. Comet assays demonstrated that DNA repair was delayed in cells silenced for the expression of ZNF281 and treated with etoposide. Furthermore, the expression of 10 DNA damage response genes was downregulated in cells treated with etoposide and silenced for ZNF281. In line with this finding, XRCC2 and XRCC4, two genes that take part in homologous recombination and non-homologous end joining, respectively, were transcriptionally activated by ZNF281 through a DNA-binding-dependent mechanism, as demonstrated by luciferase assays and Chromatin crosslinking ImmunoPrecipitation experiments. c-Myc, which also binds to the promoters of XRCC2 and XRCC4, was unable to promote their transcription or to modify ZNF281 activity. Of interest, bioinformatic analysis of 1971 breast cancer patients disclosed a significant correlation between the expression of ZNF281 and that of XRCC2. In summary, our data highlight, for the first time, the involvement of ZNF281 in the cellular response to genotoxic stress through the control exercised on the expression of genes that act in different repair mechanisms.

c-myb down regulation is associated with apoptosis in human neuroblastoma cells.

The c-myb transcriptional regulator plays a crucial role in the control of several proliferative/differentiative processes in haematopoietic cells. Its expression and function is not lineage-restricted, since c-myb is also expressed in solid tumours such as neuroblastomas, where its transcription is decreased by retinoic acid. In response to retinoic acid, neuroblastomas differentiate either towards a neuronal phenotype or undergo apoptosis. The temporal relationship between reduction in c-myb mRNA levels and the differentiative/proliferative/apoptotic processes suggests that c-myb may play a key role in the control of growth of these neuroectodermal tumours. Transfection of neuroblastoma cells with expression vectors containing segments of human c-myb cDNA in antisense orientation yielded fewer transfectant clones, with a far slower proliferation rate, than transfection with the corresponding sense construct. The dramatic growth arrest and reduction in cell number in the antisense transfectants is due to the induction of apoptosis. Apoptosis in the c-myb antisense-transfected cells is further increased in reduced serum conditions.

Cyclin D1-dependent regulation of B-myb activity in early stages of neuroblastoma differentiation.

Levels of the transcription factor B-myb must be down-regulated to allow terminal differentiation of neuroectodermal cells and yet its constitutive expression induces early markers of neural differentiation. Thus, we investigated potential mechanisms of enhanced B-myb activity in early stages of neural differentiation. We report here that B-myb expression does not decrease, cyclin A and Sp1 levels remain constant while p21 levels increase continuously upon retinoic acid-induced differentiation of the LAN-5 neuroblastoma cell line. In contrast, cyclin D1 expression is down-regulated at the onset of the differentiative process by protein destabilization. Luciferase assays of promoter activity indicate that B-myb-dependent transactivation is enhanced in LAN-5 cells treated with retinoic acid (RA) for 24 h. The enhancement is independent from cyclin A but is suppressed by a degradation-resistant mutant form of cyclin D1. The importance of cyclin D1 in controlling B-myb activity is further suggested by co-immunoprecipitation experiments, showing that the amount of cyclin D1 co-immunoprecipitated with B-myb decreased after RA treatment. Thus, B-myb may play an active role in the early stages of differentiation when its transactivation activity is enhanced as a consequence of cyclin D1 down-modulation.

Neuroblastoma specific effects of DR-nm23 and its mutant forms on differentiation and apoptosis.

DR-nm23 belongs to a gene family which includes nm23-H1, originally identified as a candidate metastasis suppressor gene. Nm23 genes are expressed in different tumor types where their levels have been alternatively associated with reduced or increased metastatic potential. Nm23-H1, -H2, DR-nm23 and nm23-H4 all possess NDP kinase activity. Overexpression of DR-nm23 inhibits differentiation and promotes apoptosis in hematopoietic cells. By contrast, it induces morphological and biochemical changes associated with neural differentiation in neuroblastoma cells. In this study, we show that mutations in the catalytic domain and in the serine 61 phosphorylation site, possibly required for protein-protein interactions, impair the ability of DR-nm23 to induce neural differentiation. Moreover, neuroblastoma cells overexpressing wild-type or mutant DR-nm23 are less sensitive to apoptosis triggered by serum withdrawal. By subcellular fractionation, wild-type and mutant DR-nm23 localize in the cytoplasm and prevalently in the mitochondrial fraction. In co-immunoprecipitation experiments, wild-type DR-nm23 binds other members of nm23 family, but mutations in the catalytic and in the RGD domains and in serine 61 inhibit the formation of hetero-multimers. Thus, the integrity of the NDP kinase activity and the presence of a serine residue in position 61 seem essential for the ability of DR-nm23 to trigger differentiation and to bind other Nm23 proteins, but not for the anti-apoptotic effect in neuroblastoma cells. These studies underline the tissue specificity of the biological effects induced by DR-nm23 expression.

The RB-related gene Rb2/p130 in neuroblastoma differentiation and in B-myb promoter down-regulation.

The retinoblastoma family of nuclear factors is composed of RB, the prototype of the tumour suppressor genes and of the strictly related genes p107 and Rb2/p130. The three genes code for proteins, namely pRb, p107 and pRb2/p130, that share similar structures and functions. These proteins are expressed, often simultaneously, in many cell types and are involved in the regulation of proliferation and differentiation. We determined the expression and the phosphorylation of the RB family gene products during the DMSO-induced differentiation of the N1E-115 murine neuroblastoma cells. In this system, pRb2/p130 was strongly up-regulated during mid-late differentiation stages, while, on the contrary, pRb and p107 resulted markedly decreased at late stages. Differentiating N1E-115 cells also showed a progressive decrease in B-myb levels, a proliferation-related protein whose constitutive expression inhibits neuronal differentiation. Transfection of each of the RB family genes in these cells was able, at different degrees, to induce neuronal differentiation, to inhibit [3H]thymidine incorporation and to down-regulate the activity of the B-myb promoter.

N-myc genomic content and DNA ploidy in stage IVS neuroblastoma.

DNA ploidy and N-myc genomic content were analyzed in a series of stage IVS neuroblastomas by flow cytometry and Southern blot hybridization, respectively. Of the 12 stage IVS neuroblastomas studied, nine were aneuploid (DNA index [DI] greater than 1), two were diploid (DI = 1), and one was not assessable for DNA content due to insufficient tumor material. N-myc gene amplification was present in two of 12 tumors. None of the aneuploid tumors exhibited N-myc amplification. Among the aneuploid neuroblastomas, the DIs were between 1.27 and 1.60, ie, in the near-triploid range. The follow-up from diagnosis ranged from 1 to 41 months (mean, 20 months). The nine neuroblastomas with near-triploid DNA content were free of disease at the end of the follow-up period. In contrast, a rapid and fatal tumor progression was observed for the three neuroblastomas with N-myc amplification and/or diploidy. Although involving only a limited series, these results strongly suggest that the combined analysis of DNA ploidy and N-myc genomic content could predict clinical outcome in stage IVS neuroblastoma and should help to identify patients for whom a more aggressive therapy is required.

Neuroblastoma: oncogenic mechanisms and therapeutic exploitation of necroptosis.

Neuroblastoma (NB) is the most common extracranial childhood tumor classified in five stages (1, 2, 3, 4 and 4S), two of which (3 and 4) identify chemotherapy-resistant, highly aggressive disease. High-risk NB frequently displays MYCN amplification, mutations in ALK and ATRX, and genomic rearrangements in TERT genes. These NB subtypes are also characterized by reduced susceptibility to programmed cell death induced by chemotherapeutic drugs. The latter feature is a major cause of failure in the treatment of advanced NB patients. Thus, proper reactivation of apoptosis or of other types of programmed cell death pathways in response to treatment is relevant for the clinical management of aggressive forms of NB. In this short review, we will discuss the most relevant genomic rearrangements that define high-risk NB and the role that destabilization of p53 and p73 can have in NB aggressiveness. In addition, we will propose a strategy to stabilize p53 and p73 by using specific inhibitors of their ubiquitin-dependent degradation. Finally, we will introduce necroptosis as an alternative strategy to kill NB cells and increase tumor immunogenicity.

Molecular cloning and sequence analysis of a cDNA coding for the mouse alpha-like embryonic globin chain x.

Cytoplasmic poly(A)+mRNA from 12-day mouse-yolk-sac erythroid cells has been used to prepare a cDNA library in the plasmid pBR322. One clone containing sequences coding for the alpha-like embryonic globin chain x, pHE52, has been identified by hybrid selection and in vitro translation of the complementary mRNA. The nucleotide sequence of pHE52 confirms that it codes for an embryonic alpha-like globin chain. The insert sequence is 316 nucleotides long, contains the codons corresponding to amino acid residues 43-141, and extends into the 3' untranslated region. An analysis of the nucleotide sequence of pHE52 and the other known alpha globins suggests that the adult-embryonic divergence began approx. 400 million years ago reflecting a difference in the evolutionary history of the alpha- and beta-globin gene complexes.

Association of near-diploid DNA content and N-myc amplification in neuroblastomas.

Seventeen neuroblastomas at different clinical stages were analysed for their N-myc copy number and flow cytometrically determined DNA content. Aneuploidy was found in 11 patients (65 per cent), whereas the remaining were near-diploid. N-myc amplification was found significantly (P less than 0.05) confined to near-diploid tumors (3 out of 6 cases). This finding indicates a very selective mechanism of oncogene amplification which is independent of gross chromosomal imbalance and limited to specific loci in the human genome. Association of near-diploidy and age at diagnosis older than 24 months was also demonstrated (P less than 0.05). Thus, flow cytometric analysis of DNA content together with N-myc gene dosage allowed us to distinguish two different subsets of neuroblastoma tumors: the first one aneuploid, with single-copy N-myc, usually observed in patients younger than 24 months with localized or IV-S clinical stages; the second one near-diploid, with frequent N-myc amplification, usually observed in patients older than 24 months with advanced clinical stages.

Transcriptional pattern of 21-hydroxylase gene (P-450C21) during embryonic development, before, and after birth in mice as determined by in situ hybridization.

21-Hydroxylase is a member of the P-450 superfamily of genes involved in the biosynthesis of cortisol and aldosterone in the adrenal cortex. Congenital adrenal hyperplasia, a well-characterized disease, originates from a lack of this enzyme. We present in this report an in situ hybridization study aimed at detecting 21-hydroxylase activity during murine development, from mid gestation to adulthood. Our results demonstrate that even during the embryonic period the adrenal cortex is the only major site of transcription of this enzyme, which is detectable beginning at embryonic day 14. In addition, a peculiar topographical pattern of transcriptional activity, characteristic of the stage of differentiation of the gland, could be drawn. Using a computer-assisted method, we were able to quantitate the relative transcription level at each stage of development. A steady increase in the level of transcription was demonstrated throughout embryonic life to birth, with a drop during the prepubertal period and a final rise at adult age. The possible physiological significance of our findings is discussed.

Transcription of N-myc and proliferation-related genes is linked in human neuroblastoma.

A definite association between the transcription of N-myc oncogene and proliferation-related genes, histone H3, c-myc and p53, was found in a set of 12 primary untreated neuroblastomas and a metastasis of one of these at relapse. Molecular analysis allowed us to discriminate between actually proliferating and non-proliferating tumors, and suggested a link between N-myc and proliferation. Flow cytometric analysis of DNA distribution was less reliable for assessing tumor proliferative activity. Our data also seem to indicate a down-regulation of c-myc by N-myc in human neuroblastoma.

Cytologic evidence for increased rRNA gene activity in hemin-induced K562(S) cells.

Hemin-induced K562(S) cells have been studied for the following parameters: cell proliferation, erythroid induction, hemoglobin accumulation, and activation of ribosomal gene clusters 48 hr after hemin induction. Increased transcriptional activity of rRNA genes has been demonstrated by cytochemical methods at both the cell population and single cell level. The following results have been obtained: (a) The vast majority of induced cells shows a highly significant increase in the number of active rRNA gene clusters per cell. At this time, the number of benzidine-positive cells and the quantity of hemoglobin per cell are almost doubled. (b) Specific rRNA gene clusters are activated within single cells. Activation can be visualized at the single gene cluster level. (c) The increase in the average number of active ribosomal gene clusters per cell is not due to clonal selection, but rather to diffuse activation of several gene clusters. (d) The transcriptional activity of rRNA genes has been shown to be regulated at the cellular level by an agent known to specifically induce derepression of genes responsible for erythroid differentiation.

Flow cytometric and molecular analysis of proliferative activity and DNA content in neuroblastoma: presence of stationary cells in S-phase.

Twenty-nine previously untreated neuroblastomas were analyzed for DNA content and percentage of cells in S-phase, both determined by flow cytometry, and N-myc oncogene copy number. Twelve of them were also tested for histone H3 transcript levels as a marker of actual proliferative activity. A significantly higher S-phase fraction was associated with advanced stages of disease, unfavorable (i.e., near-diploid and near-tetraploid) DNA content, and N-myc amplification. The occurrence of six tumors with a remarkable (greater than or equal to 10%) S-phase fraction but lacking histone H3 transcripts suggests the presence of stationary S-phase cells in neuroblastoma.