Falsely decreased ferritin concentrations in two patients with haemophagocytic lymphohistiocytosis: A case report.

The high-dose hook effect, or prozone effect, can lead to negative or falsely lowered plasma ferritin results. Here, cases of a 16-year-old boy and a 70-year-old woman with haemophagocytic lymphohystiocytosis with extremely high concentrations of plasma ferritin (387,000 µg/L and 138,000 µg/L, respectively) are presented. In both cases, falsely lowered ferritin results were reported without any analyser flag. This article emphasizes the importance of recognition of the high-dose hook effect, since a watertight solution is lacking.

Mitochondrial reactive oxygen species are scavenged by Cockayne syndrome B protein in human fibroblasts without nuclear DNA damage.

Cockayne syndrome (CS) is a human DNA repair-deficient disease that involves transcription coupled repair (TCR), in which three gene products, Cockayne syndrome A (CSA), Cockayne syndrome B (CSB), and ultraviolet stimulated scaffold protein A (UVSSA) cooperate in relieving RNA polymerase II arrest at damaged sites to permit repair of the template strand. Mutation of any of these three genes results in cells with increased sensitivity to UV light and defective TCR. Mutations in CSA or CSB are associated with severe neurological disease but mutations in UVSSA are for the most part only associated with increased photosensitivity. This difference raises questions about the relevance of TCR to neurological disease in CS. We find that CSB-mutated cells, but not UVSSA-deficient cells, have increased levels of intramitochondrial reactive oxygen species (ROS), especially when mitochondrial complex I is inhibited by rotenone. Increased ROS would result in oxidative damage to mitochondrial proteins, lipids, and DNA. CSB appears to behave as an electron scavenger in the mitochondria whose absence leads to increased oxidative stress. Mitochondrial ROS, however, did not cause detectable nuclear DNA damage even when base excision repair was blocked by an inhibitor of polyADP ribose polymerase. Neurodegeneration in Cockayne syndrome may therefore be associated with ROS-induced damage in the mitochondria, independent of nuclear TCR. An implication of our present results is that mitochondrial dysfunction involving ROS has a major impact on CS-B pathology, whereas nuclear TCR may have a minimal role.

Conceptual developments in the causes of Cockayne syndrome.

Cockayne syndrome is an autosomal recessive disease that covers a wide range of symptoms, from mild photosensitivity to severe neonatal lethal disorder. The pathology of Cockayne syndrome may be caused by several mechanisms such as a DNA repair deficiency, transcription dysregulation, altered redox balance and mitochondrial dysfunction. Conceivably each of these mechanisms participates during a different stage in life of a Cockayne syndrome patient. Endogenous reactive oxygen is considered as an ultimate cause of DNA damage that contributes to Cockayne syndrome pathology. Here we demonstrate that mitochondrial reactive oxygen does not cause detectable nuclear DNA damage. This observation implies that a significant component of Cockayne syndrome pathology may be due to abnormal mitochondrial function independent of nuclear DNA damage. The source of nuclear DNA damage to central nervous system tissue most likely occurs from extrinsic neurotransmitter signaling.

Dysmyelination not demyelination causes neurological symptoms in preweaned mice in a murine model of Cockayne syndrome.

Cockayne syndrome (CS) is a rare autosomal recessive neurodegenerative disease that is associated with mutations in either of two transcription-coupled DNA repair genes, CSA or CSB. Mice with a targeted mutation in the Csb gene (Cs-b(m/m)) exhibit a milder phenotype compared with human patients with mutations in the orthologous CSB gene. Mice mutated in Csb were crossed with mice lacking Xpc (Xp-c(-/-)), the global genome repair gene, to enhance the pathological symptoms. These Cs-b(m/m).Xp-c(-/-) mice were normal at birth but exhibited progressive failure to thrive, whole-body wasting, and ataxia and died at approximately postnatal day 21. Characterization of Cs-b(m/m).Xp-c(-/-) brains at postnatal stages demonstrated widespread reduction of myelin basic protein (MBP) and myelin in the sensorimotor cortex, the stratum radiatum, the corpus callosum, and the anterior commissure. Quantification of individual axons by electron microscopy showed a reduction in both the number of myelinated axons and the average diameter of myelin surrounding the axons. There were no significant differences in proliferation or oligodendrocyte differentiation between Cs-b(m/m).Xp-c(-/-) and Cs-b(m/+).Xp-c(-/-) mice. Rather, Cs-b(m/m).Xp-c(-/-) oligodendrocytes were unable to generate sufficient MBP or to maintain the proper myelination during early development. Csb is a multifunctional protein regulating both repair and the transcriptional response to reactive oxygen through its interaction with histone acetylase p300 and the hypoxia-inducible factor (HIF)1 pathway. On the basis of our results, combined with that of others, we suggest that in Csb the transcriptional response predominates during early development, whereas a neurodegenerative response associated with repair deficits predominates in later life.

Functional relevance of the histone gammaH2Ax in the response to DNA damaging agents.

The phosphorylation of H2Ax on its S139 site, γH2Ax, is important during DNA double-strand repair and is considered necessary for assembly of repair complexes, but its functional role after other kinds of DNA damage is less clear. We have measured the survival of isogenic mouse cell lines with the H2Ax gene knocked out, and replaced with wild-type or mutant (S139A) H2Ax genes, exposed to a range of agents with varied mechanisms of DNA damage. Knockout and mutant cells were sensitive to γ-rays, etoposide, temozolamide, and endogenously generated reactive oxygen species, each of which can include double-strand breaks among their spectra of DNA lesions. The absence or mutation of H2Ax had no influence on sensitivity to cisplatin or mitomycin C. Although UV light induced the highest levels of γH2Ax, mutation of S139 had no influence on UV sensitivity or the UV DNA damage response. Complete loss of H2Ax reduced the survival of cells exposed to UV light and reduced pChk1 induction, suggesting that sites other than S139 may impact the ATR-pChk1 pathway. The relative intensity of γH2Ax measured in Western blots in wild-type cells did not correlate with the functional importance of γH2Ax. The use of γH2Ax as a general biomarker of DNA damage is therefore potentially misleading because it is not an unambiguous indicator of double-strand breaks, and a significant fraction of DNA repair, especially involving nucleotide excision or crosslink repair, can occur without functional involvement of γH2Ax.

The DNA damage-binding protein XPC is a frequent target for inactivation in squamous cell carcinomas.

XPC, the main damage-recognition protein responsible for nucleotide excision repair of UVB damage to DNA, is lost or mutated in xeroderma pigmentosum group C (XP-C), a rare inherited disease characterized by high incidence and early onset of non-melanoma and melanoma skin cancers. The high incidence of skin cancers in XP-C patients suggests that loss of expression of XPC protein might also provide a selective advantage for initiation and progression of similar cancers in non XP-C patients in the general population. To test whether XPC is selectively lost in squamous cell carcinomas from non XP-C patients, we examined XPC expression by immunohistochemistry on a tissue microarray with 244 tissue cores, including in situ and invasive squamous-cell carcinomas (SCCs), keratoacanthoma (KA), and normal skin samples from both immunocompetent and immunosuppressed patients. We found that XPC expression was lost in 49% of invasive squamous cell carcinomas from immunocompetent patients and 59% from immunosuppressed patients. Loss of expression was correlated with deletions of chromosomal 3p and mutations in the XPC gene. The XPC gene is consequently inactivated or lost in almost half of squamous cell carcinomas from non XP-C patients. Loss or mutation of XPC may be an early event during skin carcinogenesis that provides a selective advantage for initiation and progression of squamous cell carcinomas in non XP-C patients.

A minority of foci or pan-nuclear apoptotic staining of gammaH2AX in the S phase after UV damage contain DNA double-strand breaks.

UV irradiation induces histone variant H2AX phosphorylated on serine 139 (gammaH2AX) foci and high levels of pan-nuclear gammaH2AX staining without foci, but the significance of this finding is still uncertain. We examined the formation of gammaH2AX and 53BP1 that coincide at sites of double-strand breaks (DSBs) after ionizing radiation. We compared UV irradiation and treatment with etoposide, an agent that causes DSBs during DNA replication. We found that during DNA replication, UV irradiation induced at least three classes of gammaH2AX response: a minority of gammaH2AX foci colocalizing with 53BP1 foci that represent DSBs at replication sites, a majority of gammaH2AX foci that did not colocalize with 53BP1 foci, and cells with high levels of pan-nuclear gammaH2AX without foci of either gammaH2AX or 53BP1. Ataxia-telangiectasia mutated kinase and JNK mediated the UV-induced pan-nuclear gammaH2Ax, which preceded and paralleled UV-induced S phase apoptosis. These high levels of pan-nuclear gammaH2AX were further increased by loss of the bypass polymerase Pol eta and inhibition of ataxia-telangiectasia and Rad3-related, but the levels required the presence of the damage-binding proteins of excision repair xeroderma pigmentosum complementation group A and C proteins. DSBs, therefore, represent a small variable fraction of UV-induced gammaH2AX foci dependent on repair capacity, and they are not detected within high levels of pan-nuclear gammaH2AX, a preapoptotic signal associated with ATM- and JNK-dependent apoptosis during replication. The formation of gammaH2AX foci after treatment with DNA-damaging agents cannot, therefore, be used as a direct measure of DSBs without independent corroborating evidence.

MSX1 induces the Wnt pathway antagonist genes DKK1, DKK2, DKK3, and SFRP1 in neuroblastoma cells, but does not block Wnt3 and Wnt5A signalling to DVL3.

Neuroblastoma is the most common extra-cranial solid childhood cancer; it arises from neural crest-derived cells of the sympathetic nervous system. The anomalous regulation of embryonic developmental pathways like Delta-Notch and Wnt has been implicated in aberrant cell growth and differentiation in many (childhood) tumours. We have previously found regulation of Delta-Notch pathway genes by the MSX1 neural crest development gene in a neuroblastoma cell line, and significant correlations between these genes in neuroblastic tumours. However, a clear role for the Wnt pathway in neuroblastic tumours has not yet been determined. We now analyze the complete spectrum of genes regulated by inducible expression of MSX1 in the SJNB8 neuroblastoma cell line using Affymetrix expression profiling. We show that MSX1 induces the expression of four different Wnt pathway inhibitor genes: Dickkopf 1-3 (DKK1-3) and secreted frizzled-related protein 1 (SFRP1), and provide evidence that high expression of two of these genes correlates with good prognosis. We were able to demonstrate that both the canonical Wnt3 and the alternative Wnt5A ligands are highly expressed in neuroblastic tumours and cell lines, and specifically activate the DVL3 Wnt co-receptor protein in SJNB8 neuroblastoma cells. These results suggest involvement of MSX1 in Wnt signalling and demonstrate activity of the more upstream Wnt pathway in neuroblastic cells.

Disorders of nucleotide excision repair: the genetic and molecular basis of heterogeneity.

Mutations in genes on the nucleotide excision repair pathway are associated with diseases, such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy, that involve skin cancer and developmental and neurological symptoms. These mutations cause the defective repair of damaged DNA and increased transcription arrest but, except for skin cancer, the links between repair and disease have not been obvious. Widely different clinical syndromes seem to result from mutations in the same gene, even when the mutations result in complete loss of function. The mapping of mutations in recently solved protein structures has begun to clarify the links between the molecular defects and phenotypes, but the identification of additional sources of clinical variability is still necessary.

The MSX1 homeobox transcription factor is a downstream target of PHOX2B and activates the Delta-Notch pathway in neuroblastoma.

Neuroblastoma is an embryonal tumour of the peripheral sympathetic nervous system (SNS). One of the master regulator genes for peripheral SNS differentiation, the homeobox transcription factor PHOX2B, is mutated in familiar and sporadic neuroblastomas. Here we report that inducible expression of PHOX2B in the neuroblastoma cell line SJNB-8 down-regulates MSX1, a homeobox gene important for embryonic neural crest development. Inducible expression of MSX1 in SJNB-8 caused inhibition of both cell proliferation and colony formation in soft agar. Affymetrix micro-array and Northern blot analysis demonstrated that MSX1 strongly up-regulated the Delta-Notch pathway genes DLK1, NOTCH3, and HEY1. In addition, the proneural gene NEUROD1 was down-regulated. Western blot analysis showed that MSX1 induction caused cleavage of the NOTCH3 protein to its activated form, further confirming activation of the Delta-Notch pathway. These experiments describe for the first time regulation of the Delta-Notch pathway by MSX1, and connect these genes to the PHOX2B oncogene, indicative of a role in neuroblastoma biology. Affymetrix micro-array analysis of a neuroblastic tumour series consisting of neuroblastomas and the more benign ganglioneuromas showed that MSX1, NOTCH3 and HEY1 are more highly expressed in ganglioneuromas. This suggests a block in differentiation of these tumours at distinct developmental stages or lineages.

MEIS homeobox genes in neuroblastoma.

The common pediatric tumor neuroblastoma originates from primitive neural crest-derived precursor cells of the peripheral nervous system. Neuroblastoma especially affects very young children, and can already be present at birth. Its early onset and cellular origin predict the involvement of developmental control genes in neuroblastoma etiology. These genes are indispensable for the tight regulation of normal embryonic development but as a consequence cause cancer and congenital diseases upon mutation or aberrant expression. To date however, the connotation of these genes in neuroblastoma pathogenesis is scant. This review recapitulates data on the MEIS homeobox control genes in cancer and focuses on neuroblastoma.

Characterization of plasmid pRT1 from Pyrococcus sp. strain JT1.

We discovered a 3,373-bp plasmid (pRT1) in the hyperthermophilic archaeon Pyrococcus sp. strain JT1. Two major open reading frames were identified, and analysis of the sequence revealed some resemblance to motifs typically found in plasmids that replicate via a rolling-circle mechanism. The presence of single-stranded DNA replication intermediates of pRT1 was detected, confirming this mode of replication.