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.

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.

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.

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.

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.

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.

Nucleotide excision repair gene (ERCC1) deficiency causes G(2) arrest in hepatocytes and a reduction in liver binucleation: the role of p53 and p21.

A wide range of DNA lesions, both UV and chemically induced, are dealt with by the nucleotide excision repair (NER) pathway. Defects in NER result in human syndromes such as xeroderma pigmentosum (XP), where there is a 1000-fold increased incidence of skin cancer. The ERCC1 protein is essential for NER, but ERCC1 knockout mice are not a model for XP. In the absence of exogenous DNA-damaging agents, these mice are runted and die before weaning, with dramatically accelerated liver polyploidy and elevated levels of p53. Here we present a morphological, immunological, and molecular study to understand the mechanism for the unusual liver pathology in ERCC1-deficient mice. We show that the enlarged ERCC1-deficient hepatocytes are arrested in G(2) and that DNA replication and the normal process of binucleation are both reduced. This is associated with a p53-independent increase in expression of the cyclin-dependent kinase inhibitor p21. The most dramatic feature of the ERCC1-deficient liver phenotype, the accelerated polyploidy, is not rescued by p53 deficiency, but we show that p53 is responsible for the reduced DNA replication and binucleation. We consider that the liver phenotype is a response to unrepaired endogenous DNA damage, which may reflect an additional non-NER-related function for the ERCC1 protein.

A single amino acid alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy.

A mutation equivalent to P102L in the human PrP gene, associated with Gerstmann-Straussler syndrome (GSS), has been introduced into the murine PrP gene by gene targeting. Mice homozygous for this mutation (101LL) showed no spontaneous transmissible spongiform encephalopathy (TSE) disease, but had incubation times dramatically different from wild-type mice following inoculation with different TSE sources. Inoculation with GSS produced disease in 101LL mice in 288 days. Disease was transmitted from these mice to both wild-type (226 days) and 101LL mice (148 days). In contrast, 101LL mice infected with ME7 had prolonged incubation times (338 days) compared with wild-type mice (161 days). The 101L mutation does not, therefore, produce any spontaneous genetic disease in mice but significantly alters the incubation time of TSE infection. Additionally, a rapid TSE transmission was demonstrated despite extremely low levels of disease-associated PrP.

Ataxia in prion protein (PrP)-deficient mice is associated with upregulation of the novel PrP-like protein doppel.

The novel locus Prnd is 16 kb downstream of the mouse prion protein (PrP) gene Prnp and encodes a 179 residue PrP-like protein designated doppel (Dpl). Prnd generates major transcripts of 1.7 and 2.7 kb as well as some unusual chimeric transcripts generated by intergenic splicing with Prnp. Like PrP, Dpl mRNA is expressed during embryogenesis but, in contrast to PrP, it is expressed minimally in the CNS. Unexpectedly, Dpl is upregulated in the CNS of two PrP-deficient (Prnp(0/0)) lines of mice, both of which develop late-onset ataxia, suggesting that Dpl may provoke neurodegeneration. Dpl is the first PrP-like protein to be described in mammals, and since Dpl seems to cause neurodegeneration similar to PrP, the linked expression of the Prnp and Prnd genes may play a previously unrecognized role in the pathogenesis of prion diseases or other illnesses.

Efficient BLG-Cre mediated gene deletion in the mammary gland.

Using the phage P1-derived Cre/loxP recombination system, we have developed a strategy for efficient mammary tissue specific inactivation of floxed genes. Transgenic mice were generated which express Cre DNA-recombinase under the control of the mammary gland specific promoter of the ovine beta-lactoglobulin (BLG) gene. To test the specificity of Cre mediated recombination, we crossed these mice to animals harbouring a floxed DNA ligase I allele. We show that the BLG-Cre construct specifies mammary specific gene deletion, and furthermore that it is temporally regulated, predominantly occurring during lactation. We fully characterised the extent of gene deletion in one line (line 74). In this strain the virgin gland is characterised by low levels (7%) of Cre mediated deletion, whereas 70-80% of cells within the lactating mammary gland have undergone recombination. Immunohistochemistry and indirect in situ PCR were used respectively to demonstrate that both Cre protein and Cre activity were evenly distributed throughout the population of secretory epithelial cells. The level of background recombination in non-mammary tissues was found to be < or = 1.1%, irrespective of mammary gland developmental status. Crossing the transgenic BLG-Cre strain described here to mice harbouring other floxed alleles will facilitate the functional analysis of those genes during differentiation and development of the mammary gland.

Lessons from keratin 18 knockout mice: formation of novel keratin filaments, secondary loss of keratin 7 and accumulation of liver-specific keratin 8-positive aggregates.

Here, we report on the analysis of keratin 18 null mice. Unlike the ablation of K8, which together with K18 is expressed in embryonic and simple adult epithelia, K18 null mice are viable, fertile, and show a normal lifespan. In young K18 null mice, hepatocytes were completely devoid of keratin filaments. Nevertheless, typical desmosomes were formed and maintained. Old K18 null mice, however, developed a distinctive liver pathology with abnormal hepatocytes containing K8-positive aggregates. These stained positively for ubiquitin and MM120-1 and were identified as Mallory bodies, one hallmark of human alcoholic hepatitis. This is the first demonstration that the ablation of one keratin leads to the accumulation of its single partner. Another striking finding was the absence or drastic down regulation of K7 in several tissues despite its ongoing transcription. Moreover, K18 null mice revealed new insights in the filament-forming capacity of the tail-less K19 in vivo. Due to the unexpected secondary loss of K7, only K8/19 are expressed in the uterine epithelium of K18 null mice. Immunoelectron microscopy of this tissue demonstrated the presence of typical K8/19 IF, thus highlighting in vivo that K19 is a fully competent partner for K8.

Mice with gene targetted prion protein alterations show that Prnp, Sinc and Prni are congruent.

Classical genetic analysis has identified Sinc/Prni as the major gene controlling mouse scrapie incubation time. Sinc/Prni is linked to Prnp, the gene encoding the prion protein (PrP). Prnp alleles express distinct PrP protein variants, PrP A and PrP B, which arise from codon 108L/F and 189 T/V dimorphisms. Prnp genotype segregates with incubation time length which suggests, but does not prove, that incubation time is controlled by PrP dimorphisms, and that the Sinc/Prni and Prnp loci are congruent. We have used gene targetting to construct mice in which the endogenous Prnp allele has been modified to express PrP B instead of PrP A. Challenge with a mouse-adapted BSE strain results in dramatically shortened incubation times and demonstrates that PrP dimorphisms at codon 108 and/or 189 control incubation time, and that Sinc/Prni and Prnp are congruent.

Cells from ERCC1-deficient mice show increased genome instability and a reduced frequency of S-phase-dependent illegitimate chromosome exchange but a normal frequency of homologous recombination.

The ERCC1 protein is essential for nucleotide excision repair in mammalian cells and is also believed to be involved in mitotic recombination. ERCC1-deficient mice, with their extreme runting and polyploid hepatocyte nuclei, have a phenotype that is more reminiscent of a cell cycle arrest/premature ageing disorder than the classic DNA repair deficiency disease, xeroderma pigmentosum. To understand the role of ERCC1 and the link between ERCC1-deficiency and cell cycle arrest, we have studied primary and immortalised embryonic fibroblast cultures from ERCC1-deficient mice and a Chinese hamster ovary ERCC1 mutant cell line. Mutant cells from both species showed the expected nucleotide excision repair deficiency, but the mouse mutant was only moderately sensitive to mitomycin C, indicating that ERCC1 is not essential for the recombination-mediated repair of interstrand cross links in the mouse. Mutant cells from both species had a high mutation frequency and the level of genomic instability was elevated in ERCC1-deficient mouse cells, both in vivo and in vitro. There was no evidence for an homologous recombination deficit in ERCC1 mutant cells from either species. However, the frequency of S-phase-dependent illegitimate chromatid exchange, induced by ultra violet light, was dramatically reduced in both mutants. In rodent cells the G1 arrest induced by ultra violet light is less extensive than in human cells, with the result that replication proceeds on an incompletely repaired template. Illegitimate recombination, resulting in a high frequency of chromatid exchange, is a response adopted by rodent cells to prevent the accumulation of DNA double strand breaks adjacent to unrepaired lesion sites on replicating DNA and allow replication to proceed. Our results indicate an additional role for ERCC1 in this process and we propose the following model to explain the growth arrest and early senescence seen in ERCC1-deficient mice. In the absence of ERCC1, spontaneously occurring DNA lesions accumulate and the failure of the illegitimate recombination process leads to the accumulation of double strand breaks following replication. This triggers the p53 response and the G2 cell cycle arrest, mediated by increased expression of the cyclin-dependent kinase inhibitor p21(cip1/waf1). The increased levels of unrepaired lesions and double strand breaks lead to an increased mutation frequency and genome instability.

Stability of HPRT marker gene expression at different gene-targeted loci: observing and overcoming a position effect.

For sophisticated gene targeting procedures requiring two sequential selective steps to operate efficiently it is essential that the marker genes used are not prone to position effects. The double replacement gene targeting procedure, to produce mice with subtle gene alterations, is based on the use of hypoxanthine phosphoribosyltransferase ( HPRT) minigenes in HPRT-deficient embryonic stem cells. Our standard HPRTminigene, under the control of the mouse phosphoglycerate kinase-1 gene promoter, was stably expressed at five of six target loci examined. At the remaining locus, DNA ligase I (Lig1), expression of this minigene was highly unstable. A different minigene, under the control of the mouse HPRT promoter and embedded in its natural CpG-rich island, overcame this position effect and was stably expressed when targeted to the identical site in the Lig1 locus. The promoter region of the stably expressed minigene remained unmethylated, while the promoter of the unstably expressed minigene rapidly became fully methylated. The difference in the stability of HPRT minigene expression at the same target locus can be explained in the context of the different lengths of their CpG-rich promoter regions with associated transcription factors and a resulting difference in their susceptibility to DNA methylation, rather than by differences in promoter strength.

Transgenic analysis of prion diseases.

Prion diseases are fatal transmissible neurological disorders afflicting a range of mammalian species. Although still controversial, a large body of data suggests that the causative agent may be composed entirely of a small glycoprotein. The brains of infected animals have accumulations of a pathogenic protease-resistant isoform (PrPsc) of a normal host-encoded glycoprotein, PrPc or prion protein. A number of lines of biochemical evidence implicate the disease-specific isoform, PrPsc, as the transmissible agent and genetic analysis has shown tight linkage between PrP gene mutations and polymorphisms and differential susceptibility to prion diseases, Perhaps the strongest evidence for a protein-only model of the agent is that PrP gene-ablated mice are resistant to scrapie and that mice with PrP mutation, corresponding to those found in a human familial prion disease, spontaneously develop a transmissible prion disease. This review describes the critical role that transgenic technology has played in the study of the biology of prion diseases and considers the issues raised by this work.