Daniele Castiglia

Quality of life in patients with epidermolysis bullosa

Background  Epidermolysis bullosa (EB) is a rare, inherited group of disorders characterized by blistering of the skin following friction or mechanical trauma. EB has a clinical and socioeconomic impact on patients and their families.

H1° and H3.3B mRNA levels in developing rat brain

Two overlapping rat cDNAs, covering a continuous region of 1107 base pairs, have been isolated and sequenced. The clones contain identical open reading frames, encoding a 136 amino acid long polypeptide which exhibits 100% identity to other mammalian H3.3 histone variants. We show that the inserts derive, in particular, from the H3.3B gene. We used these inserts and an insert from an H1° encoding clone, previously described (6), as probes to study the accumulation of mRNAs encoding the corresponding histone replacement variants (namely, H1° and H3.3) during rat brain development. We found that the concentration of both H1° and H3.3B mRNAs decreases from the embryonal day 18 (E18) to the postnatal day 10 (P10), with inverse correlation to protein accumulation.

Concomitant activation of Wnt pathway and loss of mismatch repair function in human melanoma

Constitutive activation of the Wnt pathway plays a key role in the development of colorectal cancer and has also been implicated in the pathogenesis of other malignancies. Deregulation of Wnt signaling mainly occurs through genetic alterations of APC, the β‐catenin gene (CTNNB1), AXIN1 and AXIN2, leading to stabilization of β‐catenin. Physiologically, AXIN2 is transcriptionally induced on Wnt signaling activation and acts as a negative feedback regulator of the pathway. In colorectal cancer, mutations in CTNNB1 and AXIN2 occur preferentially in tumors with inactivation of the mismatch repair (MMR) genes MSH2, MLH1, or PMS2. In this study, the expression of β‐catenin and AXIN2, and the mutational status of CTNNB1, APC, and AXIN2 were evaluated in two MMR‐deficient (PR‐Mel and MR‐Mel) and seven MMR‐proficient human melanoma cell lines. Only PR‐Mel and MR‐Mel cells showed nuclear accumulation of β‐catenin and expression of the AXIN2 gene, and hence, constitutive activation of Wnt signaling. Mutational analysis identified a somatic heterozygous missense mutation in CTNNB1 exon three and a germline heterozygous deletion within AXIN2 exon seven in PR‐Mel cells, and a somatic biallelic deletion within APC in MR‐Mel cells. Deregulation of Wnt signaling and a defective MMR system were also present in the original tumor of PR and MR patients. Thus, we describe additional melanomas with mutations in CTNNB1 and APC, identify for the first time a germline AXIN2 mutation in a melanoma patient and suggest that inactivation of the MMR system and deregulation of the Wnt/β‐catenin signaling pathway cooperate to promote melanoma development and/or progression.

Posttranscriptional regulation of H1° and H3.3B histone genes in differentiating rat cortical neurons

Accumulation of mRNAs encoding H1° and H3.3, two histone replacement variants, was studied in differentiating cortical neurons, cultured in a serum-free medium, with or without triiodothyronine (T3) supplementation. We found that the levels of both H1° and H3.3B mRNAs decrease inisolated neurons between the 2nd and 5th day of culture to the same extent as in vivo. At the same time, an active synthesis of the corresponding proteins was evidenced. The effects of transcription inhibition by actinomycin D and the results of nuclear run-on experiments suggest that H1° and H3.3 expression is regulated mainly at the posttranscriptional level. Concerning T3, only marginal effects were noticed, apart from up-regulation of both histone mRNAs at 2 days in culture. We propose one model for posttranscriptional regulation of the analyzed genes and discuss potential relationships to remodelling of chromatin.

The cyclin-dependent kinase inhibitor PHA-848125 suppresses the in vitro growth of human melanomas sensitive or resistant to temozolomide, and shows synergistic effects in combination with this triazene compound

PHA-848125 is a novel cyclin-dependent kinase inhibitor under Phase I/II clinical investigation. In this study, we describe, for the first time, the effect of PHA-848125 on human melanoma cells in vitro. Seven melanoma cell lines with different sensitivity to temozolomide (TMZ) were exposed to PHA-848125 for 5 days and then assayed for cell growth. In all cases, including TMZ-resistant cells, PHA-848125 IC(50) values were significantly below the maximum plasma concentrations achievable in the clinic. In the most PHA-848125-sensitive cell line, the drug caused a concentration-dependent G(1) arrest. PHA-848125 also impaired phosphorylation of the retinoblastoma protein at CDK2 and CDK4 specific sites, decreased retinoblastoma protein and cyclin A levels, and increased p21(Cip1), p27(Kip1) and p53 expression. Combined treatment with fixed ratios of TMZ plus PHA-848125 was studied in three melanoma cell lines. PHA-848125 was added to the cells 48 h after TMZ and cell growth was evaluated after 3 additional days of culture. Parallel experiments were performed in the presence of O(6)-benzylguanine (BG), to prevent repair of methyl adducts at O(6)-guanine induced by TMZ. Drug combination of TMZ plus BG and PHA-848125 produced additive or synergistic effects on cell growth, depending on the cell line. In the absence of BG, the combination was still more active than the single agents in the cell line moderately sensitive to TMZ, but comparable to PHA-848125 alone in the two TMZ-resistant cell lines. When TMZ plus BG were used in combination with PHA-848125 against cultured normal melanocytes, neither synergistic nor additive antiproliferative effects were observed. Our results indicate that PHA-848125 can have a therapeutic potential in melanoma patients, alone or combined with TMZ. Moreover this agent appears to be particularly attractive on the bases of its effectiveness against TMZ-resistant melanoma cells.

Bazex-Dupré-Christol syndrome: An ectodermal dysplasia with skin appendage neoplasms

Bazex-Dupré-Christol syndrome is a rare X-linked genodermatosis characterized by early-onset nonmelanoma skin cancers, atrophoderma follicularis, hypotrichosis, hypohidrosis, and multiple milia. Its molecular basis remains unknown and nosologic classification is debated. We report a 5-year-old child presenting sparse hair, reduced sweating, ice-pick skin depressions of the dorsum of hands, facial and limb milia, perianal skin hyperpigmentation, and hyperpigmented papules of the axillae and neck. His mother showed similar features but lacked hair involvement. Histologic examination of a skin papule obtained from the index case revealed features consistent with trichoepithelioma. Our findings indicate that trichoepitheliomas are an early sign of Bazex-Dupré-Christol syndrome and may guide the diagnosis even before the development of basal cell carcinomas. The high frequency of hypotrichosis, hypohidrosis and dry skin in Bazex-Dupré-Christol syndrome indicates that it may be better classified as an ectodermal dysplasia. Comparison with other conditions combining features of ectodermal dysplasia and proneness to skin tumors suggests the involvement of a common pathogenic pathway implicated in both skin development and cancer.

Expression of synapsin I gene in primary cultures of differentiating rat cortical neurons

Synapsin I is a neuron-specific protein which is present in two isoforms, Ia and Ib. In the last few years this protein has been demonstrated to play a central role in the regulation of neurotransmitter release and synaptic plasticity. In this paper the developmental expression of this protein has been investigated in primary neuronal cultures from fetal rat brain cortices. The presence of thyroid hormone in the culture medium stimulates an early expression of the protein without exerting any effect at the level of mRNA transcription and accumulation. These observations implicate a T3-dependent regulation of this neuron-specific gene at the level of mRNA translation.

Triiodothyronine-induced shortening of chromatin repeat length in neurons cultured in a chemically defined medium.

Abstract: At the time of terminal differentiation, mammalian cortical neurons undergo a dramatic change in the structural organization of their chromatin: the nucleosomal repeat length shortens from ∼200 base pairs in fetuses to a value of 165 base pairs after birth. These events occur several days after the end of neuronal proliferation. Previously, we reported that rat cortical neurons cultured in a very selective synthetic medium were not yet programmed to these events at the end of mitotic cycles. Herein, we report that addition of triiodothyronine to neuronal cultures induces a shortening of the chromatin repeat length comparable to the natural one.

Molecular Testing in Epidermolysis Bullosa

The development of DNA technology and improved knowledge of the structure and function of the human genome have led to the identification of the causative genes responsible for the different forms of epidermolysis bullosa (EB) and provided the opportunity to determine the precise location and type of mutations present in EB patients, allowing diagnosis of this disease at the level of the defective gene itself. The large genetic heterogeneity of EB, however, precludes the direct use of molecular testing for EB diagnosis. In addition, only a few diagnostic or research laboratories in the world are equipped to perform mutational screening, which is still labor intensive and associated with considerable costs, because most mutations are unique to one or a few families. This article reviews the most popular methods used in EB molecular analysis.

Family Burden in Epidermolysis Bullosa is High Independent of Disease Type/Subtype

Epidermolysis bullosa is a rare, inherited group of disorders characterized by blistering of the skin following friction or mechanical trauma. The aim of this study was to assess the family burden of epidermolysis bullosa in children aged 0-7 years. A postal survey was conducted. The perceived severity of the disease was evaluated by the caregivers, using the Patient Global Assessment 5-point scale. The caregiver received the Family Strain Questionnaire and the 12-item General Health Questionnaire to assess the probable presence of depression/anxiety. A single-item analysis was also performed for questions related to the burden of disease. Forty-two families were invited to participate. Data from 28 young patients and their caregivers were analysed (response rate 66.7%). The family burden increased with increasing caregivers perceived disease severity, with increasing patients body surface involved, and if parents had depression/anxiety, reaching statistical significance in several Family Strain Questionnaire scales. The family burden due to epidermolysis bullosa is very high independent of disease type/subtype.