DNA repair investigations in nine Italian patients affected by trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by brittle hair, mental and growth retardation, peculiar face, ichthyosis, and in 20% of the reported cases photosensitivity. Cellular photosensitivity due to the same genetic defect present in xeroderma pigmentosum group D (XP-D) has been described in several patients. Nine patients with clinical symptoms diagnostic for TTD have been identified in Italy to date. We report the results of DNA repair investigations performed in cultured fibroblasts from these patients and 8 TTD parents. Survival, DNA repair synthesis and RNA synthesis following UV irradiation were all normal in the 8 TTD heterozygous cell strains. Among the 9 TTD-affected individuals, normal cellular UV sensitivity was observed in the 2 patients without signs of clinical photosensitivity. In contrast, the other 7 TTD cell strains showed a notable reduction in UV-induced DNA repair synthesis (UDS) levels, ranging between 40% and 5-15% of normal values. Complementation analysis indicated that in the repair-deficient TTD cell strains the genetic defect is the same as that present in XP-D cells. The biochemical heterogeneity of the XP-D defect in TTD patients characterized by different degrees of defective UDS results in different patterns of response to the killing effect of UV light in non-proliferating cells.     

Defects in the DNA repair and transcription gene ERCC2(XPD) in trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by brittle hair with reduced sulfur content, ichthyosis, peculiar face, and mental and growth retardation. Clinical photosensitivity is present in approximately 50% of TTD patients but is not associated with an elevated frequency of cancers. Previous complementation studies show that the photosensitivity in nearly all of the studied patients is due to a defect in the same genetic locus that underlies the cancer-prone genetic disorder xeroderma pigmentosum group D (XP-D). Nucleotide-sequence analysis of the ERCC2 cDNA from three TTD cell strains (TTD1V1, TTD3VI, and TTD1RO) revealed mutations within the region from amino acid 713-730 and within previously identified helicase functional domains. The various clinical presentations and DNA repair characteristics of the cell strains can be correlated with the particular mutations found in the ERCC2 locus. Mutations of Arg658 to either His or Cys correlate with TTD cell strains with intermediate UV-sensitivity, mutation of Arg722 to Trp correlates with highly UV-sensitive TTD cell strains, and mutation of Arg683 to Trp correlates with XP-D. Alleles with mutation of Arg616 to Pro or with the combined mutation of Leu461 to Val and deletion of 716-730 are found in both XP-D and TTD cell strains.     

Complementation studies in cells from patients affected by trichothiodystrophy with normal or enhanced UV photosensitivity

A normal level of UV-induced DNA-repair synthesis (UDS) was observed in fibroblasts from a patient affected by trichothiodystrophy (TTD) without photosensitivity. This finding indicates that the hypersensitivity to UV light and the reduced UDS due to the presence of xeroderma pigmentosum complementation group D mutation (XP-D), described in photosensitive TTD patients, are not constantly associated with TTD. Complementation analysis in heterokaryons, obtained by fusion of repair-proficient with repair-deficient TTD cells, demonstrates that cells from the patient showing normal photosensitivity are able to restore UDS in UV-hypersensitive TTD cells.     

UV damage causes uncontrolled DNA breakage in cells from patients with combined features of XP-D and Cockayne syndrome

Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. Defects in NER result in three different human disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). Two cases with the combined features of XP and CS have been assigned to the XP-D complementation group. Despite their extreme UV sensitivity, these cells appeared to incise their DNA as efficiently as normal cells in response to UV damage. These incisions were, however, uncoupled from the rest of the repair process. Using cell-free extracts, we were unable to detect any incision activity in the neighbourhood of the damage. When irradiated plasmids were introduced into unirradiated XP-D/CS cells, the ectopically introduced damage triggered the induction of breaks in the undamaged genomic DNA. XP-D/CS cells thus have a unique response to sensing UV damage, which results in the introduction of breaks into the DNA at sites distant from the damage. We propose that it is these spurious breaks that are responsible for the extreme UV sensitivity of these cells.     

A new nucleotide-excision-repair gene associated with the disorder trichothiodystrophy

The sun-sensitive, cancer-prone genetic disorder xeroderma pigmentosum (XP) is associated in most cases with a defect in the ability to carry out excision repair of UV damage. Seven genetically distinct complementation groups (i.e., A–G) have been identified. A large proportion of patients with the unrelated disorder trichothiodystrophy (TTD), which is characterized by hair-shaft abnormalities, as well as by physical and mental retardation, are also deficient in excision repair of UV damage. In most of these cases the repair deficiency is in the same complementation group as is XP group D. We report here on cells from a patient, TTD1BR, in which the repair defect complements all known XP groups (including XP-D). Furthermore, microinjection of various cloned human repair genes fails to correct the repair defect in this cell strain. The defect in TTD1BR cells is therefore in a new gene involved in excision repair in human cells. The finding of a second DNA repair gene that is associated with the clinical features of TTD argues strongly for an involvement of repair proteins in hair-shaft development.     

Expression of the cDNA for the beta subunit of human casein kinase II confers partial UV resistance on xeroderma pigmentosum cells.

An immortalized xeroderma pigmentosum cell line belonging to the complementation group D (XP-D) was transfected with a normal human cDNA clone library constructed in a mammalian expression vector. Following UV-irradiation-selection, a transformant having a stable, partially UV-resistant phenotype was isolated. A transfected cDNA of partial length was rescued from the transformant's cellular DNA by in vitro amplification, using expression-vector specific oligonucleotides as primers in a polymerase chain reaction (PCR). Expression of this cDNA complemented the UV sensitivity of the XP-D cell line to the UV-resistance levels characteristic of the primary transformant. The nucleotide sequence of the cDNA was determined. The deduced protein identified the cDNA as encoding for the beta subunit of casein kinase II (CKII-beta). Similar to the effect exerted by the truncated CKII-beta cDNA, expression of a cDNA clone encompassing the complete translated region of CKII-beta leads to XP-D cells partially resistant to UV-irradiation. However, transfection of CKII-beta cDNA could also partially complement the UV-sensitivity of a xeroderma pigmentosum cell line belonging to group C (XP-C). Analysis by Southern, Northern and RNAase mismatch cleavage techniques did not reveal any functional defect in the CKII-beta gene of cell lines derived from either 7 XP-D or 10 XP-C families. We therefore consider it unlikely that either the XP-D or the XP-C DNA repair deficiency is associated with a defect in the beta subunit of casein kinase II. Nevertheless, our findings suggest the possibility that the cell's response to DNA damage is modulated by CKII-dependent protein phosphorylation.     

Measurement of bacterial volume by transmission-through-dye imaging

Transmission-through-dye (TTD) microscopy makes possible direct measurement of bacterial volume, irrespective of cell shape. The technique can be realized on any brightfield microscope and is applicable to bacteria of all shapes. TTD imaging requires that intact bacteria be immobilized on a flat transparent surface, such as a glass coverslip.     

Measurement of bacterial volume by transmission-through-dye imaging

Transmission-through-dye (TTD) microscopy makes possible direct measurement of bacterial volume, irrespective of cell shape. The technique can be realized on any brightfield microscope and is applicable to bacteria of all shapes. TTD imaging requires that intact bacteria be immobilized on a flat transparent surface, such as a glass coverslip.     

Impact of Response Shift on Time to Deterioration in Quality of Life Scores in Breast Cancer Patients

Background

This prospective multicenter study aimed to study the impact of the recalibration component of response-shift (RS) on time to deterioration (TTD) in health related quality of life (QoL) scores in breast cancer (BC) patients and the influence of baseline QoL expectations on TTD.

Methods

The EORTC-QLQ-C30 and BR-23 questionnaires were used to assess the QoL in a prospective multicenter study at inclusion (T0), at the end of the first hospitalization (T1) and, three (T2) and 6 months after the first hospitalization (T3). Recalibration was investigated by the then-test method. QoL expectancy was assessed at diagnosis. Deterioration was defined as a 5-point decrease in QoL scores, considered a minimal clinically important difference (MCID). TTD was estimated using the Kaplan-Meier method. Cox regression analyses were used to identify factors influencing TTD.

Results

From February 2006 to February 2008, 381 women were included. Recalibration of breast cancer patients'' internal standards in the assessment of their QoL had an impact on TTD. Median TTD were significantly shorter when recalibration was not taken into account than when recalibration was taken into account for global health, role-functioning, social-functioning, body-image and side effects of systemic therapy. Cox multivariate analyses showed that for body image, when recalibration was taken into account, radiotherapy was associated with a shorter TTD (HR: 0.60[0.38–0.94], whereas, no significant impact of surgery type on TTD was observed. For global health, cognitive and social functioning dimensions, patients expecting a deterioration in their QoL at baseline had a significantly shorter TTD.

Conclusions

Our results showed that RS and baseline QoL expectations were associated with time to deterioration in breast cancer patients.     

Nucleotide excision repair and cancer

Nucleotide excision repair (NER) is the most versatile and best studied DNA repair system in humans. NER can repair a variety of bulky DNA damages including UV-light induced DNA photoproducts. NER consists of a multistep process in which the DNA lesion is recognized and demarcated by DNA unwinding. Then, a ~28 bp DNA damage containing oligonucleotide is excised followed by gap filling using the undamaged DNA strand as a template. The consequences of defective NER are demonstrated by three rare autosomal-rezessive NER-defective syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). XP patients show severe sun sensitivity, freckling in sun exposed skin, and develop skin cancers already during childhood. CS patients exhibit sun sensitivity, severe neurologic abnormalities, and cachectic dwarfism. Clinical symptoms of TTD patients include sun sensitivity, freckling in sun exposed skin areas, and brittle sulfur-deficient hair. In contrast to XP patients, CS and TTD patients are not skin cancer prone. Studying these syndromes can increase the knowledge of skin cancer development including cutaneous melanoma as well as basal and squamous cell carcinoma in general that may lead to new preventional and therapeutic anticancer strategies in the normal population.     

Xeroderma pigmentosum and the role of UV-induced DNA damage in skin cancer

Xeroderma pigmentosum (XP) is a rare, autosomal recessive disease that is characterized by the extreme sensitivity of the skin to sunlight. Compared to normal individuals, XP patients have a more than 1000-fold increased risk of developing cancer on sun-exposed areas of the skin. Genetic and molecular analyses have revealed that the repair of ultraviolet (UV)-induced DNA damage is impaired in XP patients owing to mutations in genes that form part of a DNA-repair pathway known as nucleotide excision repair (NER). Two other diseases, Cockayne syndrome (CS) and the photosensitive form of trichothiodystrophy (TTD), are linked to a defect in the NER pathway. Strikingly, although CS and TTD patients are UV-sensitive, they do not develop skin cancer. The recently developed animal models that mimic the human phenotypes of XP, CS and TTD will contribute to a better understanding of the etiology of these diseases and the role of UV-induced DNA damage in the development of skin cancer.     

Nucleotide Excision Repair Disorders and the Balance Between Cancer and Aging

Cancer incidence increases with age and is driven by accumulation of mutations in the DNA. In many so-called premature aging disorders, cancer appears earlier and at elevated rates. These diseases are predominantly caused by genome instability and present with symptoms, including cancer, resembling “segments” of aging and are thus often referred to as “segmental progerias”. Two related segmental progerias, Cockayne syndrome (CS) and trichothiodystrophy (TTD), don’t fit this pattern. Although caused by defects in genome maintenance via the nucleotide excision DNA repair (NER) pathway and displaying severe progeroid symptoms, CS and TTD patients appear to lack any cancer predisposition. More strikingly, genetic defects in the same NER pathway, and in some cases even within the same gene, XPD, can also give rise to disorders with greatly elevated cancer rates but without progeria (xeroderma pigmentosum). In this review, we will discuss the connection between genome maintenance, aging and cancer in light of a new mouse model of XPD disease.