Dosimetric Effect of Biozorb Markers for Accelerated Partial Breast Irradiation in Proton Therapy.

PURPOSE: To investigate dosimetric implications of biodegradable Biozorb (BZ) markers for proton accelerated partial breast irradiation (APBI) plans. MATERIALS AND METHODS: Six different BZs were placed within in-house breast phantoms to acquire computed tomography (CT) images. A contour correction method with proper mass density overriding for BZ titanium clip and surrounding tissue was applied to minimize inaccuracies found in the CT images in the RayStation planning system. Each breast phantom was irradiated by a monoenergetic proton beam (103.23 MeV and 8×8 cm2) using a pencil-beam scanning proton therapy system. For a range perturbation study, doses were measured at 5 depths below the breast phantoms by using an ionization chamber and compared to the RayStation calculations with 3 scenarios for the clip density: the density correction method (S1: 1.6 g/cm3), raw CT (S2), and titanium density (S3: 4.54 g/cm3). For the local dose perturbation study, the radiographic EDR2 film was placed at 0 and 2 cm below the phantoms and compared to the RayStation calculations. Clinical effects of the perturbations were retrospectively examined with 10 APBI plans for the 3 scenarios (approved by our institutional review board). RESULTS: In the range perturbation study, the S1 simulation showed a good agreement with the chamber measurements, while excess pullbacks of 1∼2 mm were found in the S2 and S3 simulations. The film study showed local dose shadowing and perturbation by the clips that RayStation could not predict. In the plan study, no significant differences in the plan quality were found among the 3 scenarios. However, substantial range pullbacks were observed for S3. CONCLUSION: The density correction method could minimize the dose and range difference between measurement and RayStation prediction. It should be avoided to simply override the known physical density of the BZ clips for treatment planning owing to overestimation of the range pullback.

Differential expression of microRNAs during melanoma progression: miR-200c, miR-205 and miR-211 are downregulated in melanoma and act as tumour suppressors.

BACKGROUND: The incidence of malignant melanoma is increasing faster than that for any other cancer. Histological examination of skin excision biopsies remains the standard method for melanoma diagnosis and prognosis. Significant morphological overlap between benign and malignant lesions complicates diagnosis, and tumour thickness is not always an accurate predictor of prognosis. METHODS: To identify improved molecular markers to support histological examination, we used microarray analysis of formalin-fixed and paraffin-embedded samples from different stages of melanomagenesis to identify differentially expressed microRNAs (miRNAs). Differential expression was validated by qRT-PCR, and functional studies were carried out after transfection of miRNA precursors or inhibitors into melanoma cells to modulate miRNA expression. RESULTS: In all, 20 miRNAs showed highly significant differential expression between benign naevi and either primary or metastatic melanomas, the majority being downregulated in melanoma, whereas only 2 miRNAs, namely miR-203 and miR-205, were differentially expressed between primary and metastatic melanomas. In functional in vitro assays, overexpression of miR-200c and miR-205 inhibited anchorage-independent colony formation and overexpression of miR-211 inhibited both anchorage-independent colony formation and invasion. CONCLUSION: We have identified a series of differentially expressed miRNAs that could be useful as diagnostic or prognostic markers for melanoma and have shown that three miRNAs (namely miR-200c, miR-205 and miR-211) act as tumour suppressors.

Cisplatin regulates the MAPK kinase pathway to induce increased expression of DNA repair gene ERCC1 and increase melanoma chemoresistance.

The incidence of malignant melanoma is growing rapidly worldwide and there is still no effective therapy for metastatic disease. Melanoma is the second most common cancer among young adults in the UK, where incidence rates have more than quadrupled since the 1970s. Increased expression of a number of DNA repair genes has been reported in melanoma and this likely contributes to its extreme resistance to conventional DNA-damaging chemotherapeutics. One such chemotherapeutic that is effective against a range of other cancers, but not melanoma, is cisplatin. The DNA repair proteins ERCC1 and XPF are needed to remove cisplatin-induced DNA damage and we have investigated the response of these proteins to cisplatin in melanoma. The expression of both genes is induced by cisplatin. Use of a MEK inhibitor showed that ERCC1, but not XPF induction was regulated by the mitogen-activated protein kinase (MAPK) pathway, with reduction in expression of DUSP6, the phosphatase that inactivates the extracellular signal-regulated kinase (ERK), being particularly important. DUSP6 overexpression prevented cisplatin induction of both ERCC1 and XPF, resulting in increased sensitivity to cisplatin. A novel ERCC1 mRNA was found that initiated upstream of the normal transcription initiation site, and was strongly regulated by both cisplatin and the MAPK pathway and its role in cisplatin resistance merits further study. The cisplatin induction of ERCC1 and XPF provides important insights into the resistance of melanoma to DNA-damaging chemotherapeutics, which is one of the major obstacles to melanoma treatment.

Using stem cells to study and possibly treat type 1 diabetes.

Stem cells with the potential to form many different cell types are actively studied for their possible use in cell replacement therapies for several diseases. In addition, the differentiated derivatives of stem cells are being used as reagents to test for drugs that slow or correct disease phenotypes found in several degenerative diseases. This paper explores these approaches in the context of type 1 or juvenile diabetes, pointing to recent successes as well as the technical and theoretical challenges that lie ahead in the path to new treatments and cures.

A novel transcript for DNA repair gene Ercc1 in mouse skin.

The nucleotide excision repair pathway deals with UV-induced DNA damage. The tissue that receives by far the greatest exposure to UV is the skin and we have investigated the possibility that expression of the nucleotide excision repair gene, Ercc1, may display different properties in the skin to deal with a more demanding role in that tissue. ERCC1, in a complex with XPF, is the structure--specific endonuclease responsible for incising 5' to the UV-induced lesion. We identified a novel Ercc1 mRNA in mouse skin that originates from an alternative upstream promoter. Levels of this skin-specific transcript were low in embryonic skin and increased rapidly after birth, but there was no induction by UV, either in adult skin, or in a cultured keratinocyte model. Levels of the skin-specific Ercc1 transcript were higher in albino than pigmented mouse strains, but there was no difference in ERCC1 protein levels and the expression of the skin-specific transcript was found to be determined by the Ercc1 gene sequence rather than by coat pigmentation. Using an Ercc1 transgene the promoter for the skin-specific transcript was mapped to a region around 400 bp upstream of the normal promoter, where a transposable element with known promoter activity was found in albino but not in pigmented strains.

Nuclear reprogramming in cells.

Nuclear reprogramming describes a switch in gene expression of one kind of cell to that of another unrelated cell type. Early studies in frog cloning provided some of the first experimental evidence for reprogramming. Subsequent procedures included mammalian somatic cell nuclear transfer, cell fusion, induction of pluripotency by ectopic gene expression, and direct reprogramming. Through these methods it becomes possible to derive one kind of specialized cell (such as a brain cell) from another, more accessible, tissue (such as skin) in the same individual. This has potential applications for cell replacement without the immunosuppression treatments that are required when cells are transferred between genetically different individuals. This article provides some background to this field, a discussion of mechanisms and efficiency, and comments on prospects for future nuclear reprogramming research.

Pathways to new beta cells.

Diabetes is a leading health problem of the world and its prevalence continues to rise. With Type I diabetes, and in some patients with Type II, the lack of insulin can be counterbalanced by providing new beta (insulin-producing) cells. For Type I diabetes, treating the autoimmune attack remains a serious challenge. Several strategies to produce new beta cells have been proposed. These include differentiation from embryonic stem cells, proliferation of existing adult beta cells, derivation from putative adult progenitors/stem cells, and reprogramming of non-beta cells to beta cells. Each of these strategies has distinct merits and risks, and they are at different stages of understanding and development. In particular, the approach based on differentiation from embryonic stem cells has had strong support and in recent years has made notable progress. Nevertheless, significant hurdles remain to transform the current research into future therapies. To expedite this transformation, we believe particular emphasis should be placed on overcoming key knowledge gaps in beta-cell biology, developing strategies that produce patient-specific beta cells, and carefully addressing potential treatment-related complications or limitations.

Mice with skin-specific DNA repair gene (Ercc1) inactivation are hypersensitive to ultraviolet irradiation-induced skin cancer and show more rapid actinic progression.

Ercc1 has an essential role in the nucleotide excision repair (NER) pathway that protects against ultraviolet (UV)-induced DNA damage and is also involved in additional repair pathways. The premature death of simple Ercc1 mouse knockouts meant that we were unable to study the role of Ercc1 in the skin. To do this, we have used the Cre-lox system to generate a skin-specific Ercc1 knockout. With a Cre transgene under control of the bovine keratin 5 promoter we achieved 100% recombination of the Ercc1 gene in the epidermis. Hairless mice with Ercc1-deficient skin were hypersensitive to the short-term effects of UV irradiation, showing a very low minimal erythemal dose and a dramatic hyperproliferative response. Ultraviolet-irradiated mice with Ercc1-deficient skin developed epidermal skin tumours much more rapidly than controls. These tumours appeared to arise earlier in actinic progression and grew more rapidly than tumours on control mice. These responses are more pronounced than have been reported for other NER-deficient mice, demonstrating that Ercc1 has a key role in protecting against UV-induced skin cancer.

Lack of involvement of nucleotide excision repair gene polymorphisms in colorectal cancer.

DNA repair has an essential role in protecting the genome from damage by endogenous and environmental agents. Polymorphisms in DNA repair genes and differences in repair capacity between individuals have been widely documented. For colorectal cancer, the loss of mismatch repair gene activity is a key genetic determinant. Nucleotide excision repair (NER), recombination repair (RR) and base excision repair (BER) pathways have critical roles in protection against other cancers, and we wished to investigate their role in colorectal cancer. We have compared the frequency of polymorphisms in the NER genes, XPD, XPF, XPG, ERCC1; in the BER gene, XRCC1; and in the RR gene, XRCC3; in colorectal cancer patients and in a control group. No significant associations were found for any of the NER gene polymorphisms or for the XRCC1 polymorphism. The C allele (position 18067) of the XRCC3 gene was weakly but significantly associated with colorectal cancer (odds ratio 1.52, 95% confidence interval 1.04-2.22, P=0.03). For all patients who were heterozygous for any of the repair genes studied, tumour tissue was investigated for loss of heterozygosity (LOH). Only one example of LOH was found for all the genes examined. From the association and LOH data, we conclude that these genes do not have an important role in protection against colorectal carcinogenesis.

Correction of liver dysfunction in DNA repair-deficient mice with an ERCC1 transgene.

The ERCC1 gene is essential for the repair of UV-induced DNA damage. Unlike most genes in the nucleotide excision repair (NER) pathway, ERCC1 is also involved in recombinational repair. Perhaps for this reason, ERCC1 knockout mice are not a model for the human NER deficiency disorder, xeroderma pigmentosum. Instead, ERCC1 null mice are severely runted and die before weaning from liver failure with accelerated hepatocyte polyploidy that is more reminiscent of a premature ageing disorder. To permit study of the role of ERCC1 in other tissues we have corrected the liver ERCC1 deficiency with a transgene under the control of a liver-specific promoter. The transgene alleviated runting and extended the lifespan. The elevated level of oxidative DNA damage and premature liver polyploidy were reversed and liver function was corrected. A widespread mitochondrial dysfunction was identified and an essential role for ERCC1 in the kidney was also revealed with transgene-containing ERCC1-deficient animals going on to die of renal failure. The nuclei of kidney proximal tubule cells became polyploid in a similar way to the premature liver polyploidy observed in younger ERCC1-deficient animals. We believe that this is a response to the accumulation of endogenous DNA damage in these particularly susceptible tissues which cannot be repaired in ERCC1-deficient animals.

Induction of pancreatic differentiation by signals from blood vessels.

Blood vessels supply developing organs with metabolic sustenance. Here, we demonstrate a role for blood vessels as a source of developmental signals during pancreatic organogenesis. In vitro experiments with embryonic mouse tissues demonstrate that blood vessel endothelium induces insulin expression in isolated endoderm. Removal of the dorsal aorta in Xenopus laevis embryos results in the failure of insulin expression in vivo. Furthermore, using transgenic mice, we show that ectopic vascularization in the posterior foregut leads to ectopic insulin expression and islet hyperplasia. These results indicate that vessels not only provide metabolic sustenance, but also provide inductive signals for organ development.

Changing a single amino acid in the N-terminus of murine PrP alters TSE incubation time across three species barriers.

The PrP gene of the host exerts a major influence over the outcome of transmissible spongiform encephalopathy (TSE) disease, but the mechanism by which this is achieved is not understood. We have introduced a specific mutation into the endogenous murine PrP gene using gene targeting to produce transgenic mice with a single amino acid alteration (proline to leucine) at amino acid position 101 in their PrP protein (P101L). The effect of this alteration on incubation time, targeting and PrP(Sc) formation has been studied in TSE-infected animals. Transgenic mice carrying the P101L mutation in PrP have remarkable differences in incubation time and targeting of central nervous system pathology compared with wild-type littermates, following inoculation with infectivity from human, hamster, sheep and murine sources. This single mutation can alter incubation time across three species barriers in a strain-dependent manner. These findings suggest a critical role for the structurally 'flexible' region of PrP in agent replication and targeting of TSE pathology.

Analysis of pancreatic development in living transgenic zebrafish embryos.

Using DNA constructs containing regulatory sequences of the zebrafish Pdx-1 and insulin genes, germline transgenic zebrafish expressing the green fluorescent protein (GFP) reporter gene in the pancreas were generated. For both constructs, the GFP expression patterns in transgenic embryos were consistent with the mRNA expression patterns detected by RNA in situ hybridization. A deletion promoter analysis revealed that positive and negative cis-acting elements were involved in regulation of insulin gene expression. Three-dimensional reconstructions imaged from living embryos using two-photon laser-scanning microscopy (TPLSM) demonstrated that the zebrafish pancreas is formed from a single dorsal pancreatic cell mass. This is in contrast to mammals where the pancreas derives from both dorsal and ventral anlage. These transgenic fish should be useful for in vivo studies of factors involved in specifying and regulating pancreatic development and function.

Induction of HO-1 and NOS in doppel-expressing mice devoid of PrP: implications for doppel function.

Ectopic expression of the doppel (Dpl) protein, a homologue of the prion protein (PrP), was recently associated with cerebellar Purkinje cell degeneration observed in two aging prion protein knock-out (Prnp(0/0)) mouse lines. We investigated the possible role of Dpl in oxidative metabolism. Two Prnp(0/0) mouse lines of similar genetic background were studied. One line expresses Dpl in the brain and displays Dpl-associated cerebellar abnormalities. The other has no elevated expression of Dpl and no cerebellar abnormalities. We observed a correlation between Dpl expression and the induction of both heme oxygenase 1 (HO-1) and nitric oxide synthase systems (nNOS and iNOS). These responses are suggestive of increased oxidative stress in the brains of the Dpl-expressing Prnp(0/0) mice. No induction was observed with Hsp-60, indicating a specific response by the HO/NOS system. We proposed that Dpl expression exacerbates oxidative damage that is antagonistic to the protective function of wild-type PrP.

A one-step gene amplification system for use in cultured mammalian cells and transgenic animals.

Gene amplification is widely used for the production of pharmaceuticals and therapeutics in situations where a mammalian system is essential to synthesise a fully active product. Current gene amplification systems require multiple rounds of selection, often with high concentrations of toxic chemicals, to achieve the highest levels of gene amplification. The use of these systems has not been demonstrated in specialised mammalian cells, such as embryonic-stem cells, which can be used to generate transgenic animals. Thus, it has not yet proved possible to produce transgenic animals containing amplified copies of a gene of interest, with the potential to synthesise large amounts of a valuable gene product. We have developed a new amplification system, based around vectors encoding a partially disabled hypoxanthine phosphoribosyltransferase (HPRT) minigene, which can achieve greater than 1000-fold amplification of HPRT and the human growth hormone gene in a single step in Chinese hamster-lung cells. The amplification system also works in mouse embryonic-stem cells and we have used it to produce mice which express 30-fold higher levels of human protein C in milk than obtained with conventional transgenesis using the same protein C construct. This system should also be applicable to large animal transgenics produced by nuclear transfer from cultured cell lines.

Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes.

The mechanisms by which the epithelium of the digestive tract and its associated glands are specified are largely unknown. One clue is that several transcription factors are expressed in specific regions of the endoderm prior to and during organogenesis. Pdx-1, for example, is expressed in the duodenum and pancreas and Pdx-1 inactivation results in an arrest of pancreatic development after buds formation. Similarly, ngn3 is transiently expressed in the developing pancreas and a knockout results in the absence of endocrine cells. This paper focuses on the question of whether these and other transcription factors, known to be necessary for pancreatic development, are also sufficient to drive a program of pancreatic organogenesis. Using in ovo electroporation of chick embryos, we show that ectopic expression of Pdx-1 or ngn3 causes cells to bud out of the epithelium like pancreatic progenitors. The Pdx-1-expressing cells extinguish markers for other nonpancreatic regions of the endoderm and initiate, but do not complete, pancreatic cytodifferentiation. Ectopic expression of ngn3 is sufficient to turn endodermal cells of any region into endocrine cells that form islets expressing glucagon and somatostatin in the mesenchyme. The results suggest that simple gene combinations could be used in stem cells to achieve specific endodermal tissue differentiation.

Nucleotide excision repair gene XPD polymorphisms and genetic predisposition to melanoma.

The nucleotide excision repair pathway has evolved to deal with UV light-induced DNA damage. Individuals with the rare inherited nucleotide excision repair deficiency disease xeroderma pigmentosum have a 1000-fold increased incidence of skin cancer. We are interested in the possibility that more subtle changes in nucleotide excision repair genes, resulting in either a reduced capacity for repair or in altered interactions between repair proteins and components of the cell cycle control machinery, might constitute important genetic risk factors for the development of skin cancer in the general population. To investigate this hypothesis we have compared the frequency of polymorphisms in exons 6, 22 and 23 of the XPD gene in melanoma patients and a control group. For each of these two allele polymorphisms one of the alleles was over-represented in the melanoma group and there was a significant association with melanoma. Importantly, this association did not extend to markers immediately flanking the XPD gene, thus providing evidence that XPD gene polymorphisms might predispose to melanoma in the general population. There is a report that one of the polymorphic XPD alleles (exon 23 Lys), which is over-represented in the melanoma group, has reduced repair proficiency and we discuss the possibility that this is the causal change to the XPD gene that predisposes to melanoma.

Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells.

Human embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of in vitro fertilized human blastocysts. We examined the potential of eight growth factors [basic fibroblast growth factor (bFGF), transforming growth factor beta1 (TGF-beta1), activin-A, bone morphogenic protein 4 (BMP-4), hepatocyte growth factor (HGF), epidermal growth factor (EGF), beta nerve growth factor (betaNGF), and retinoic acid] to direct the differentiation of human ES-derived cells in vitro. We show that human ES cells that have initiated development as aggregates (embryoid bodies) express a receptor for each of these factors, and that their effects are evident by differentiation into cells with different epithelial or mesenchymal morphologies. Differentiation of the cells was assayed by expression of 24 cell-specific molecular markers that cover all embryonic germ layers and 11 different tissues. Each growth factor has a unique effect that may result from directed differentiation and/or cell selection, and we can divide the overall effects of the factors into three categories: growth factors (Activin-A and TGFbeta1) that mainly induce mesodermal cells; factors (retinoic acid, EGF, BMP-4, and bFGF) that activate ectodermal and mesodermal markers; and factors (NGF and HGF) that allow differentiation into the three embryonic germ layers, including endoderm. None of the growth factors directs differentiation exclusively to one cell type. This analysis sets the stage for directing differentiation of human ES cells in culture and indicates that multiple human cell types may be enriched in vitro by specific factors.