Development of an integrated environmental monitoring protocol for SARS-CoV-2 contamination. Applications at the IRCSS San Martino Polyclinic Hospital in Genoa, Italy.

SCIENTIFIC BACKGROUND: Environmental sampling of SARS-CoV-2 is a fundamental tool for evaluating the effectiveness of non-specific prophylaxis measures in counteracting virus spread. The purpose of our work was to evaluate the effectiveness of the different sampling methods in the hospital setting to assess their correlation with the structural, functional, and operational situation of the monitored departments and to define the dynamics of the spread of the virus in indoor environments. METHODS: The monitoring (air bubbling sampling, surface wipe test) was carried out at the San Martino Polyclinic Hospital (Genoa, Italy) in the period since April 2020 to June 2021. The presence of viral RNA in the collected samples was evaluated by qPCR. The infection capacity of the samples collected was also evaluated by an in vitro challenge test on cells sensitive to SARS-CoV-2 infection. RESULTS: The percentage of positivity with respect to the number of tests performed (sensitivity) were air bubbler 50%, wipe test 17%, and challenge test 11%. Only 20% of the samples tested positive in the wipe test and 43% of the samples tested positive in the bubbler sampling were also positive in the challenge test. All the positivity obtained was detected at a distance of less than 2 m and height of less than 1.5 from COVID-19 patients. CONCLUSIONS: Environmental contamination from SARS-CoV-2 detected at the San Martino Polyclinic Hospital is found lower than similar assessments performed in other hospitals both in Italy and abroad. Our study predicted that environmental monitoring of SARS-CoV-2 must be carried out in an integrated way by not using a single sampling method, as each individual test has a different biological significance and performance. However, the virus detected by wipe test only is often a degraded viral fragment and not an intact infecting virion.

Mutation frequencies of GNAQ, GNA11, BAP1, SF3B1, EIF1AX and TERT in uveal melanoma: detection of an activating mutation in the TERT gene promoter in a single case of uveal melanoma.

BACKGROUND: Uveal melanoma is the most frequent primary tumour of the eye. It is molecularly clearly distinct from cutaneous melanoma and shows a different pattern of driver mutations. The influence of sunlight ultraviolet (UV) exposure on the aetiology of uveal melanoma is a matter of debate. The recent identification of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene with UV-induced cytidine-to-thymidine transitions in cutaneous melanoma prompted us to investigate whether these mutations also occur in uveal melanoma. METHODS: We analysed 50 cases of uveal melanoma obtained from enucleation surgery for mutations in the genes GNAQ, GNA11, BAP1, SF3B1, EIFAX1 and TERT, measured gene expression using microarrays and analysed gene copy numbers by SNP arrays. RESULTS: We detected a TERT mutation in only one case of a 57-year-old white male patient with clinical and histopathological features typical for uveal melanoma. The tumour showed mutations in GNA11 and EIF1AX that are typical for uveal melanoma and absent from cutaneous melanoma. No mutations were detected in GNAQ, BAP1 and SF3B1 that are frequently mutated in uveal melanoma. Both copies of chromosome 3 were retained. Several tumours among which the one carrying the TERT promoter mutation showed elevated TERT expression. Consistent with previous reports, GNAQ is inversely associated with chromosome 3 monosomy and metastasis. BAP1 mutations are significantly associated with chromosome 3 monosomy but not with relapse. CONCLUSION: These data indicate that TERT mutations are rare in uveal melanoma. No conclusion can be drawn on their potential influence on tumour progression.

Endocrine disruptor agent nonyl phenol exerts an estrogen-like transcriptional activity on estrogen receptor positive breast cancer cells.

Several substances widely dispersed in the environment including hormones, industrial by-products and pollutants exert hormone like activity affecting steroid-responsive physiological systems. These compounds, named endocrine disruptors, are suspected to affect the mammalian reproductive system. However it is still unclear whether these substances are able to elicit estrogen like activity at the low concentrations encountered in the environment. Here we compare the effects of the endocrine disruptor nonylphenol with the effects elicited by 17-ß-estradiol on gene transcription in the human breast cancer cell line MCF7. The correlation of the nonylphenol induced gene expression alterations with a reference profile of estradiol treated cells shows that nonylphenol at a concentration of 100 nM exerts a significant effect on estrogen responsive gene transcription in MCF7 cells. Most of the genes regulated by 17-ß-estradiol respond to the nonylphenol in the same direction though to a much lesser extent. Molecular modeling of the potential interaction of nonylphenol with the estrogen receptor α shows that nonylphenol is likely to bind to the estrogen receptor α.

Exogenous hormonal regulation in breast cancer cells by phytoestrogens and endocrine disruptors.

Observations on the role of ovarian hormones in breast cancer growth, as well as interest in contraception, stimulated research into the biology of estrogens. The identification of the classical receptors ERα and ERß and the transmembrane receptor GPER and the resolution of the structure of the ligand bound to its receptor established the principal molecular mechanisms of estrogen action. The presence of estrogen-like compounds in many plants used in traditional medicine or ingested as food ingredients, phytoestrogens, as well as the estrogenic activities of many industrial pollutants and pesticides, xenoestrogens, have prompted investigations into their role in human health. Phyto- and xenoestrogens bind to the estrogen receptors with a lower affinity than the endogenous estrogens and can compete or substitute the hormone. Xenoestrogens, which accumulate in the body throughout life, are believed to increase breast cancer risk, especially in cases of prenatal and prepuberal exposure whereas the role of phytoestrogens is still a matter of debate. At present, the application of phytoestrogens appears to be limited to the treatment of post-menopausal symptoms in women where the production of endogenous estrogens has ceased. In this review we discuss chemistry, structure and classification, estrogen signaling and the consequences of the interactions of estrogens, phytoestrogens and xenoestrogens with their receptors, the complex interactions of endogenous and exogenous ligands, the evaluation of the health risks related to xenoestrogens, and the perspectives toward the synthesis of potent third generation selective estrogen receptor modulators (SERMs).

Microarray expression profiles of angiogenesis-related genes predict tumor cell response to artemisinins.

Artemisinin (ARS) and its derivatives are used for the second-line therapy of malaria infections with Plasmodium falciparum and P. vivax. ARSs also reveal profound antitumor activity in vitro and in vivo. In the present investigation, we correlated the mRNA expression data of 89 angiogenesis-related genes obtained by microarray hybridization from the database of the US National Cancer Institute with the 50% growth inhibition concentration values for eight ARSs (ARS, arteether (ARE), artesunate (ART), artemisetene, arteanuine B, dihydroartemisinylester stereoisomers 1 and 2). The constitutive expression of 30 genes correlated significantly with the cellular response to ARSs. By means of hierarchical cluster analysis and cluster image mapping expression, profiles were identified that determined significantly the cellular response to ART, ARE, artemether and dihydroartemisinylester stereoisomer 1. We have exemplarily validated the microarray data of six out of these 30 genes by real-time RT-PCR in seven cell lines. The fact that sensitivity and resistance of tumor cells could be predicted by the mRNA expression of angiogenesis-related genes indicate that ARSs reveal their antitumor effects at least in part by inhibition of tumor angiogenesis. As many chemopreventive drugs exert antiangiogenic features, ARSs might also be chemopreventive in addition to their cytotoxic effects.

Antiangiogenic activity of chemopreventive drugs.

Tumors growing within the host form dynamic aberrant tissue that consists of host components, including the stroma, an expanding vasculature and often chronic inflammation, in addition to the tumor cells themselves. These host components can contribute to, rather than limit, tumor expansion, whereas deprivation of vessel formation has the potential to confine tumors in small, clinically silent foci. Therapeutic inhibition of vessel formation could be best suited to preventive strategies aimed at the suppression of angiogenesis in primary tumors in subjects at risk, or of micrometastases after surgical removal of a primary tumor. Our analysis of potential cancer chemopreventive molecules including N-acetylcysteine, green tea flavonoids and 4-hydroxyphenyl-retinamide has identified antiangiogenic activities that could account--at least in part--for the tumor prevention effects observed with these compounds. These drugs appear to target common mechanisms of tumor angiogenesis that may permit identification of critical targets for antiangiogenic therapy and antiangiogenic chemoprevention.

Somatostatin inhibits tumor angiogenesis and growth via somatostatin receptor-3-mediated regulation of endothelial nitric oxide synthase and mitogen-activated protein kinase activities.

Somatostatin was reported to inhibit Kaposi's sarcoma (KS) cell (KS-Imm) xenografts through an antiangiogenic activity. Here, we show that somatostatin blocks growth of established KS-Imm tumors with the same efficacy as adriamycin, a clinically effective cytotoxic drug. Whereas KS-Imm cells do not express somatostatin receptors (SSTRs), endothelial cells express several SSTRs, in particular SSTR3. We investigated the molecular mechanisms and receptor specificity of somatostatin inhibition of angiogenesis. Somatostatin significantly inhibited angiogenesis in vivo in the matrigel sponge assay; this inhibition was mimicked by the SSTR3 agonist L-796778 and reversed by the SSTR3 antagonist BN81658, demonstrating involvement of SSTR3. In vitro experiments showed that somatostatin directly affected different endothelial cell line proliferation through a block of growth-factor-stimulated MAPK and endothelial nitric oxide (NO) synthase (eNOS) activities. BN81658 reversed somatostatin inhibition of cell proliferation, NO production, and MAPK activity, indicating that SSTR3 activation is required for the effects of somatostatin in vitro. Finally in vivo angiogenesis assays demonstrated that eNOS inhibition was a prerequisite for the antiangiogenic effects of somatostatin, because high concentrations of sodium nitroprusside, an NO donor, abolished the somatostatin effects. In conclusion, we demonstrate that somatostatin is a powerful antitumor agent in vivo that inhibits tumor angiogenesis through SSTR3-mediated inhibition of both eNOS and MAPK activities.

Are there CAG repeat expansion-related disorders outside the central nervous system?

Expansions of poly-glutamine tracts in proteins that are expressed in the central nervous system cause neurodegenerative diseases. The altered proteins accumulate over long periods of time, forming nuclear inclusions, and lead to neuronal cell death. A similar mechanism could also be operant in non-dividing cells outside the central nervous system because nuclear inclusions are not limited to neurons. In addition, variations of the repeat length within the normal range may affect cellular function as it has been shown for the androgen receptor that is involved in neoplastic degeneration of several tissues. We have identified a poly-glutamine/poly-proline repeat in the homeobox gene DLX6. DLX genes are expressed in non-proliferative cells of the apical ectodermal ridge of developing limbs. Ablation of these cells leads to limb malformation. We propose that CAG triplet expansions in this gene could lead to cell death in the apical ectodermal ridge causing limb malformations. Indeed, autosomal dominant limb malformations with increasing severity in successive generations have been linked to the chromosomal region that contains DLX6. The analysis of a limited number of patients affected by split hand/foot malformation so far revealed only a slight modifier effect of repeat length within the normal range and no expansions have been detected.

The coding region of the human DLX6 gene contains a polymorphic CAG/CCG repeat.

The region on chromosome 7q21-22 is frequently altered in several human neoplasias such as uterine leiomyoma, myeloid leukemia and breast cancer. The same region has also been linked to split hand/split foot malformation type 1 and to involutional osteoporosis. Our analysis of genes that map to this region has led to the identification of the so far unknown first exon of the homeobox gene DLX6, a mammalian homologue of the Drosophila distal-less gene. Distal-less is a downstream target of the trithorax transcription factors. Translocations involving the mammalian homologue of trithorax, ALL-1, leading to its constitutive activation cause leukemia. We describe here that the first exons of human and mouse DLX6 genes contain a multiple trinucleotide repeat region. We have analyzed the CAG repeat length in 90 subjects and were able to identify five alleles with 11 to 20 CAG repeats.

Somatic alterations of the androgen receptor CAG repeat in human colon cancer delineate a novel mutation pathway independent of microsatellite instability.

The human androgen receptor gene contains a polymorphic CAG repeat region ranging from 8 to about 35 repeats in the normal human population. The repeat length is inversely related to the transactivation potential of the receptor. We have analyzed the repeat length in 50 sporadic colon cancer samples in comparison to surrounding healthy mucosa and have found somatic reductions of up to 10 repeats in 5 cases (10%), 3 of which were complex, probably involving both alleles. Alterations occurred in tumors with and without microsatellite instability indicating that they follow an independent mutation pathway. The similar repeat of the huntingtin gene did not show any somatic alterations in the same cases. No correlation to sex, tumor stage, location, or histology was evident. In the tumors that showed somatic reductions, the reduced allele was present in at least half of the cells and thus in most, if not all, of the tumor component of the sample. Somatic reductions of the androgen receptor CAG repeat thus occur frequently, through a pathway distinct from microsatellite instability and early during colon carcinogenesis. The receptor is expressed in most normal and neoplastic tissue samples analyzed. Apparent growth selection of cells bearing shortened AR alleles suggests that androgens contribute to colon carcinogenesis in a yet unknown way.

Altered expression of androgen-receptor isoforms in human colon-cancer tissues.

Many groups have examined of androgen the effects on normal and neoplastic colon tissues, but no clear picture has hitherto emerged. In particular, the presence and the function of the androgen receptor (AR) has only partially been investigated in the past. The present study reports analysis of expression of the AR gene as messenger RNA and as protein in surgical samples of neoplastic colon mucosa and of corresponding healthy surrounding tissue. Specific binding for DHT, demonstrating the presence of AR, was observed in almost all the samples (2 samples out of 12 were negative). No significant difference was observed between healthy and neoplastic mucosa, or between male and female patients. A further characterization of AR was performed with Western blot, using 2 different primary antibodies. Both AR isoforms, AR-B and AR-A, were detected in healthy mucosa, while only AR-A, resolving at 87 kDa, was observed in neoplastic mucosa. RT-PCR analysis revealed the transcript for AR in both healthy and neoplastic mucosa in 10 samples; no message was detectable in 2 samples (negative also for binding); 2 additional samples presented AR mRNA only in healthy colon mucosa, 2 others only in neoplastic mucosa. In addition, a variant AR messenger RNA, probabily derived from alternative splicing, was observed. We found that AR is expressed both in healthy and in neoplastic colon mucosa, either as mRNA or as protein. Neoplastic colon tissue shows a characteristic loss of expression of the AR-B isoform, while AR-A expression is maintained. These findings underscore the possible role of androgen and its receptor in colon carcinogenesis.

Progress towards gene therapy for cancer.

This review highlights the current strategies being employed towards gene therapy of cancer. Conceptually, the most simple diseases to treat with gene therapy would be monogenic inherited diseases, such as hemophilia. However, the vast majority of current gene therapy trials are for treatment of cancer patients, due to the recognition of gene alterations in cancer and the critical need for improvement of cancer therapy. Gene-based therapies for cancer in clinical trials include strategies that involve immuno-therapy, induction of drug sensitivity in tumor cells or resistance to chemotherapy of critical host tissues, and compensation for oncosuppressor loss or ablation of oncogenes. Two broad approaches have been used to deliver DNA to cells, a series of viral vectors and the use of plasmid DNA vectors, which have different advantages with regard to efficiency of gene transfer, ease of production and safety. Examined objectively, many of the first studies in cancer gene therapy clinical trials have provided information of critical importance for the design of more efficient second-generation protocols. Gene therapy represents one of the most important developments in oncology, however, before this can be realized as standard treatment the technical problems of gene delivery and safety must be overcome. Here we focus on methods and strategies used to achieve cancer gene therapy and the current clinical trials.

Alternative splicing of the estrogen receptor primary transcript normally occurs in estrogen receptor positive tissues and cell lines.

Several laboratories have described estrogen receptor mRNA variants created by skipping internal exons. Some of the putative proteins encoded for by these variants have been functionally characterized by transfection analyses. The variant lacking exon 5 would lead, if translated, to a truncated receptor which shows dominant positive transactivation activity in the absence of hormone. It has been postulated that the variant could account for anti-estrogen resistant tumor growth and for expression of the progesterone receptor in estrogen negative tumors. In order to understand the possible role this and other variants may have in the tumorigenesis of mammary tissue we have carried out a thorough analysis of variants expressed in a tumor cell line (MCF-7), in a tumor sample and in a sample of normal breast tissue derived from mammary reduction surgery. We performed rt-PCR analyses followed by hybridization with exon specific oligonucleotide probes. By these means we have detected nine different variants co-expressed in MCF-7 cells and at least the major variants were equally expressed in normal and neoplastic breast tissue. The same is true for the variant lacking exon 5 which, however, resulted to be a variant of low expression in the three samples analyzed. Variant formation appeared to be restricted to the estrogen receptor messenger since several other members of the superfamily of nuclear receptors did not show variant formation. We also have analyzed the effect of the most abundantly expressed variant, the exon 4 lacking variant, on normal estrogen receptor function, on the growth and on the response to estradiol and to tamoxifen of MCF-7 cells. Although over-expressed at high levels this variant has, if any, only marginal effects on the expression of endogenous estrogen regulated genes and on growth and response to the hormone and its antagonist. Although the lack of function of this variant cannot be extrapolated to other variants, their involvement in tumor formation appears rather unlikely since they are also expressed in normal tissue and the single variant is expressed in addition to many others, some of which might have opposing effects. Variant formation is, however, specific for the estrogen receptor and apparently regulated with tissue specificity as our expression analysis in normal mouse tissues shows. Therefore the variants probably have a physiological significance yet to be discovered.

Growth of LNCaP human prostate cancer cells is stimulated by estradiol via its own receptor.

We report that growth of LNCaP human prostate cancer cells is significantly stimulated (up to 120% above control) by physiological estradiol (E2) concentrations. This growth increase appears to be comparable to that induced by either testosterone or dihydrotestosterone, as also reported by others. This paper presents novel illustrative evidence for estrogen-binding proteins and messenger RNA transcripts in LNCaP cells. In fact, 1) the reverse transcriptase-polymerase chain reaction system documented normal messenger RNA for estrogen receptors (ER); 2) the radioligand binding assay allowed the detection of high affinity, reduced capacity binding sites in both soluble and nuclear cell fractions; and 3) the immunocytochemical analysis showed a consistently intensive staining for both ER and progesterone receptors. Compared to other human estrogen-responsive mammary cancer cells, MCF7 and ZR75-1, ER expression in LNCaP cells was not significantly lower, as shown by levels of the ER transcripts, number of sites per cell, or femtomoles per mg DNA as well as the percentage and intensity of immunocytochemical staining. A relative estimate of ER expression obtained by matching LNCaP with another human prostate cancer cell line, PC3, always displayed significantly and consistently higher levels in LNCaP cells. The detection of relatively high type I ER content in either cell compartment of LNCaP cells was paralleled by a highly intensive staining for progesterone receptors. In addition, evidence that the synthetic androgen R1881 did not compete for type I binding of E2 and that any E2-induced growth was completely reversed by the pure antiestrogen ICI-182,780, but unaffected by the antiandrogen Casodex, clearly suggests that the biological response of LNCaP cells to E2 is mediated via its own receptor.

Coexpression of multiple estrogen receptor variant messenger RNAs in normal and neoplastic breast tissues and in MCF-7 cells.

Mammary cancers often develop into a hormone-independent and antagonist-resistant growth phase. The molecular mechanisms of this transition are not clear. Recently, it has been proposed that estrogen receptor variants derived from alternative splicing might lead to dominant positive transcription factors acting on estrogen response elements, even in the absence of the hormone. We show here the comprehensive analysis of expression of estrogen receptor variants lacking internal exons in the estrogen receptor-positive mammary carcinoma cell line MCF-7, in a tumor sample, and in healthy breast tissue taken from reduction surgery. Variants are identified by reverse transcription PCR and hybridization to exon-specific oligonucleotide probes. In MCF-7 cells we detected 10 variants including 5 that have not been described before. Skipping one, two, or three exons occurs. The major variants detected in the cell line are also present in normal and neoplastic tissues. Quantitative variations allow no conclusions of a potential involvement of the variants in neoplastic processes. Rather, the variants appear to be present normally and thus might have a physiological role. Given the expression of the variants in normal tissue, and given the expression of potentially dominant positive variants in conjunction with potentially dominant negative ones, we suggest that these variants do not account for hormone antagonist resistance.