Dr Stephen Ainger

PHD

Post Doctoral

About me

Postdoctoral Researcher

Translational Research Institute, PA Hospital, Brisbane, Australia.January 2015 – Present 

Translational research instituteSkin cell cultures, Protein analysis, UVR exposure, ligand activation, student instruction, scientific report writing, oral conference presentations, data analysis, article reviews.

Post Doctorate

Institute of Molecular Bioscience, UQ, Brisbane, Australia.May 2012 – Present (2 years 9 months)Growing monocultures of melanocytic cells (melanomas and primary melanocytes) and treating with siRNA/shRNA/lentiviruses/UVR radiation/ligands/hydrogen peroxide for changes in signalling pathways, examining cell survival and protein expression in these cells, writing scientific reports and journal articles, training students in laboratory skills, presenting work at international conferences by posters and oral presentations.

Customer Service Officer

Medicare AustraliaJanuary 2004 – February 2007 (3 years 2 months), Prahran, Melbourne, Australia.Customer service, payouts, forms, data entry, phone liaisons.

Medical Scientist

Gribbles PathologyDec 2002 – Dec 2003 (1 year 1 month), Gribbles Pathology, Bendigo, Australia.Routine human and veterinary medical science tests on animal specimens including haematology, microbiology, parasitology, biochemistry, specimen reception, patient and doctor liaisons.

Medical Scientist

Bendigo Health Care GroupMay 2001 – December 2002 (1 year 8 months), BHCG, Bendigo, Australia.Routine histology of human specimens and urgent frozen section analysis, blood bank, haematology, specimen reception, assisting procedures.

Laboratory Technician

Dorevitch PathologyMarch 1992 – April 2001 (9 years 2 months), Dorevitch Pathology, Bendigo, Australia.Routine analysis in Haematology, Biochemistry, Microbiology, but mainly in Histology sectioning, staining, assisting cut up, frozen urgent sections, specimen reception, patient and doctor liasons.

 

Projects

Myxomatosis/Cytokines & Skin Cancers/DCT in Melanoma

The role of DCT in UVR and ROS protection.

May 2010 – Present

Learning about the function dopachrome tautomerase plays in the protective roles of melanocytes in the skin via direct UVR protection from DNA damage, removal of reactive oxygen species and enhanced DNA repair.

Dr Stephen Ainger, 
Assoc. Prof. R.A.Sturm
, J. Helen Leonard
, S.S.Wong
, H.Yang,
 Dr D. Roberts
, Assoc. Prof. Brian Gabrielli
, 
Dr Duka Skalamera
.

The expression of cytokines in the progression of skin cancers

February 2007 – November 2007

Immunohistochemistry was used to examine the expression of various cytokines in normal skin, pre-malignant (benign) and skin cancers such as TNFa, IL-10, IL-8, IL-6, IFNy, IL-12.

Dr Stephen Ainger, Dr Visalini Muthusamy
, Terry Piva
, Dr. Rethika Ravi

An outbreak of myxomatosis in a local rabbit population in Northern Victoria is dependent on Mosquito transmission over the summer months.

November 1990 – November 1991

Over 1 summer season, wild rabbits were collected from a Northern Victorian site, and blood samples were taken along with data on each animal's health and fleas were collected if present. Blood was analysed for myxomatosis by ELIZA, and flea numbers counted. Mosquitoes were collected and each species characterised. Hen's egg culture was also used to detect virus in serum samples. The infected rabbits only increased during mosquito increases during summer, fleas gave a sub-lethal dose during winter.


Dr Stephen Ainger, Dr Graeme Robinson

DCT, IRF4, MC1R

Publications

Effect of MC1R variant allele status on MSH-ligand induction of dopachrome tautomerase in melanocytes co-cultured with keratinocytes.

Experimental Dermatology, 20(8): 681-684.

April 1, 2011

A co-culture system of melanocytic cells and keratinocytes was used to examine dendricity and dopachrome tautomerase (DCT) responses in low penetrant ‘r’ homozygote and ‘R ⁄ +’ heterozygote MC1R variant allele expressing cells compared to that of wild-type (WT) cells. The V60L -/- homozygote r variant cells showed similar responses to ligand as WT MC1R strains, while V92M -/- homozygote r variant cells were generally shown to have greater dendricity and express higher DCT than the WT cells, even at basal levels. The R151C +/- heterozygote cells showed similar responses to WT cells, while the R160W +/- and D294H +/- variant cells were reduced in their responses to NDP-MSH, but still had an active cAMP response with forskolin treatment. These responses are consistent with the dominant negative effect of these alleles on the MC1R WT allele that has previously been demonstrated genetically and biochemically.

Stephen A. Ainger, Shu S. Wong, Donald W. Roberts, J. Helen Leonard, Richard A. Sturm

 

MC1R Variant Allele Effects on UVR-Induced Phosphorylation of p38, p53, and DDB2 Repair Protein Responses in Melanocytic Cells in Culture.

Journal of Investigative Dermatology, 132(5): 1452-1461.

February 16, 2012

Variant alleles of the human melanocortin 1 receptor (MC1R) reduce the ability of melanocytes to produce the dark pigment eumelanin, with R alleles being most deficient. Cultured melanocytes of MC1R R/R variant genotype give reduced responses to [Nle4, D-Phe7]a-melanocyte-stimulating hormone (NDP-MSH) ligand stimulation and lower levels of DNA repair than MC1R wild-type strains. p38 controls xeroderma pigmentosum (XP)-C recruitment to DNA damage sites through regulating ubiquitylation of the DNA damage–binding protein 2 (DDB2) protein, and p53 is implicated in the nuclear excision repair process through its regulation of XP-C and DDB2 protein expression. We report the effects of MC1R ligand treatment and UVR exposure on phosphorylation of p38 and p53, and DDB2 protein expression in MC1R variant strains. Wild-type MC1R melanocyte strains grown together with keratinocytes in coculture, when treated with NDP-MSH and exposed to UVR, gave synergistic activation of p38 and p53 phosphorylation, and were not replicated by R/R variant melanocytes, which have lower basal levels of phosphorylated forms of p38. Minor increases in p38 phosphorylation status in R/R variant melanocyte cocultures could be attributed to the keratinocytes alone. We also found that MC1R wild-type strains regulate DDB2 protein levels through p38, but MC1R R/R variant melanocytes do not. This work confirms the important functional role that the MC1R receptor plays in UVR stress–induced DNA repair.

Shu Shyan Wong, Stephen Ainger
, J. Helen Leonard, Richard A. Sturm

 

Melanocortin MC1 receptor in human genetics and model systems.

European Journal of Pharmacology, 660(1): 103-110.

January 1, 2011

The melanocortin MC1 receptor is a G-protein coupled receptor expressed in the melanocytes of the skin and hair and is known for its key role in the regulation of human pigmentation. Melanocortin MC1 receptor activation after ultraviolet radiation exposure results in a switch from the red/yellow pheomelanin to the brown/black eumelanin pigment synthesis within cutaneous melanocytes; this pigment is then transferred to the surrounding keratinocytes of the skin. The increase in melanin maturation and uptake results in tanning of the skin, providing a physical protection of skin cells from ultraviolet radiation induced DNA damage. Melanocortin MC1 receptor polymorphism is widespread within the Caucasian population and some variant alleles are associated with red hair colour, fair skin, poor tanning and increased risk of skin cancer. Here we will discuss the use of mouse coat colour models, human genetic association studies, and in vitro cell culture studies to determine the complex functions of the melanocortin MC1 receptor and the molecular mechanisms underlying the association between melanocortin MC1 receptor variant alleles and the red hair colour phenotype. Recent research indicates that melanocortin MC1 receptor has many non-pigmentary functions, and that the increased risk of skin cancer conferred by melanocortin MC1 receptor variant alleles is to some extent independent of pigmentation phenotypes. The use of new transgenic mouse models, the study of novel melanocortin MC1 receptor response genes and the use of more advanced human skin models such as 3D skin reconstruction may provide key elements in understanding the pharmacogenetics of human melanocortin MC1 receptor polymorphism.

Beaumont, K. A., Wong, S. S., Ainger, S. A., Liu, Y. Y., Patel, M. P., Millhauser, G. L., Smith, J. J., Alewood, P. F., Leonard, J. H., Sturm, R. A. 

Molecular analysis of common polymorphisms within the human Tyrosinase locus and genetic association with pigmentation traits.

Pigment Cell Melanoma Res., 27(4): 552-564.

April 17, 2014

We have compared the melanogenic activities of cultured melanocytes carrying two common TYR alleles as homozygous 192S-402R wild-type, heterozygous and homozygous variant. This includes assays of TYR protein, DOPAoxidase activity, glycosylation and temperature sensitivity of protein and DOPAoxidase levels. Homozygous wild-type strains on average had higher levels of TYR protein and enzyme activity than other genotypes. Homozygous 402Q/Q melanocytes produced significantly less TYR protein, displayed altered trafficking and glycosylation, with reduced DOPAoxidase. However, near wild-type TYR activity levels could be recovered at lower growth temperature. In a sample population from Southeast Queensland, these two polymorphisms were present on four TYR haplotypes, designated as WT 192S-402R, 192Y-402R and 192S-402Q with a double-variant 192Y-402Q of low frequency at 1.9%. Based on cell culture findings and haplotype associations, we have used an additive model to assess the penetrance of the ten possible TYR genotypes derived from the combination of these haplotypes.

Jagirdar, K., Smit, D. J., Ainger, S. A., Lee, K. J., Brown, D. L., Chapman, B., Zhen Zhao, Z., Montgomery, G. W., Martin, N. G., Stow, J. L., Duffy, D. L., Sturm, R. A.

DCT protects human melanocytic cells from UVR and ROS damage and increases cell viability.

Experimental Dermatology, 23(12): 916-921.

October 20, 2014

Dopachrome tautomerase (DCT) is involved in the formation of the photoprotective skin pigment eumelanin, and has also been shown to have a role in response to apoptotic stimuli and oxidative stress. The effect of DCT on UVR DNA damage responses and survival pathways in human melanocytic cells was examined by knockdown experiments using melanoma cells, neonatal foreskin melanoblasts (MB) in monoculture and in co-culture with human keratinocytes. MB cells strains genotyped as either MC1R WT or MC1R RHC homozygotes, which are known to be deficient in DCT, were transduced with lentivirus vectors for either DCT knockdown or over-expression. We found melanoma cell survival was reduced by DCT depletion and by UVR over time. UVR-induced p53 and pp53-Ser15 levels were reduced with DCT depletion. Knockdown of DCT in MC1R WT and MC1R RHC MB cells reduced their survival after UVR exposure, whereas increased DCT protein levels enhanced survival. DCT depletion reduced p53 and pp53-Ser15 levels in WM266-4 melanoma and MC1R WT MB cells, while MC1R RHC MB cells displayed variable levels. Both MC1R WT and RHC genotypes of MB cells were responsive to UVR at 3h with increases in both p53 and pp53-Ser15 proteins. MC1R WT MB cell strains in co-culture with keratinocytes have an increased cell survival after UVR exposure when compared to those in monoculture, a protective effect which appears to be conferred by the keratinocytes.

Ainger, S. A., Yong, X. L., Wong, S. S., Skalamera, D., Gabrielli, B., Leonard, J. H., Sturm, R. A.

Research fields

Melanoma