The Hole Story:
Studies using Skin Tissue ctd
Inducing Skin Protection
By studying what happens when skin surfaces are irradiated with UV light Mason is trying to identify the chemical messengers that are involved so that they can then be manufactured and applied artificially. We know that there are a number of substances in the skin which are theoretically capable of interacting with UV radiation, by absorbing it, for instance. (One of these substances happens to be DNA, which is probably one of reasons why you get sun damage.)
The idea is to give a person a natural protective effect without having to go out in the sun to get it. Instead of having to damage yourself in order to get protection you'd be able to get the protection before you go out in the sun. This might involve some sort of drug that people could take or perhaps a cream applied to the skin. There are certain advantages in ease of use of drug that is swallowed but Mason says at this stage she has a slight preference for a locally applied material because there might also be unforeseen effects with a drug. Some of the substances that we're likely to be investigating are peptides, she says. This means they would be broken down if taken orally, so that they may not be terribly useful if they are swallowed, but we don't know for sure and there may similar substances that could be swallowed. At this stage, and it is still very early days, I would expect that these substances could perhaps be incorporated in some way into a sunscreen base and applied over a period of time. This way they could give protection while the natural protective mechanisms are being developed through exposure to the sun.
The research work that Rebecca Mason is involved in is largely done in vitro (in laboratory dishes) using cells taken from human skin samples. What she does is to grow them in culture, that is in a special mixture that encourages cell growth, and look at a number of factors that might affect their function. The skin sample is first thoroughly cleaned and sterilised with iodine solution in order to get rid of any bacterial contamination. After cleaning, a protein digester is used to allow the separation of the epidermis from the dermis, the bottom part of the skin. Then, for the pigment cell culture, the separating point between the epidermis and the dermis is scraped to get off a mixed lot of cells which includes some pigment cells, some keratinocytes and some other cells called fibroblasts. Fibroblast cells are the general connective tissue cells that make up most of the the dermis.
The task then is to separate the pigment cells from the rest.Keratinocytes don't survive very well in the material that is used for growing the pigment cells, so they are not a problem. Fibroblast contamination can still be a problem and there are a number of strategies used that helps get rid of these cells.
Keratinocytes are grown by a different method. Initially the skin is treated in the same way to sterilise it. Once the epidermis has been separated, it is cut into very small pieces, about 1 mm square cubes. It is then patted down and allowed to attach onto a plastic culture surface. Then after a little while, once the tissue has attached to the culture surface, more culture medium is added and after a few days the keratinocytes start growing out in a nice even layer. Keratinocytes. are faster growing cells than the pigment cells and this method produces as many cells as are needed. There is always a shortage of pigment cells because they are fairly slow growing cells but fortunately most of the experiments don't require a great many of them.
With the assistance of a research assistant, Nalini Dissanayake, the work so far has been to look at the effects of different parts of the solar spectrum on the activity of the cells themselves when they are irradiated. The source of UV radiation which they use can be manipulated to some extent in order to look at different parts of the spectrum. By using different filters she can look at the effects of UVB and UVA combined or just UVA or UVB on their own. The output of these sun lamps somewhat resembles that of normal sunlight in that it has got quite a sharp cut off above the range at which UV penetrates but it doesn't mimic the full spectral range of natural sunlight. It tends to be more intense than natural sunlight but approximations can be made by simply varying the time of irradiation.
When keratinocytes are irradiated in this way they produce an unknown substance, or more likely more than one substance. When these substances are put with pigment cells that have never been irradiated, the pigment cells will get a tanning type response and increase their pigmentation. Even non-irradiated keratinocytes multiply faster. So one or more of these substances are likely to be involved in the tanning and the skin thickening response to UV.
There are two alternative ways of finding out what these substances are. One is by making up a large amount of them and analysing them with a chromatograph. The other way is by trying to guess in advance what the substances are and do specific tests to confirm the guess. There are certain things Mason thinks might be involved, like a particular type of peptide, which people generally think of as coming from the pituitary gland but which in fact appear to be made in the skin as well.
She goes about these tests by using neutralising antibodies. Neutralising antibodies attach to the substance of interest and stop them carrying out their normal function or activity. (Not all antibodies do that. Some of them attach to the substance but don't stop it working.) The idea is to mix the neutralising antibody with the dish full of UV irradiated cells and see whether the antibody to a particular substance stops some of the thickening or tanning activity. If there is some evidence that there is inhibition of activity then it indicates that the particular substance may be involved in protection.
If this line of research is successful it may have some commercial value. Mason has already received some expressions of interest from cosmetic companies but because the work is still at a fairly early stage she hasn't pursued these offers. We wanted to maintain some independence, she says. They have just been expressions of interest. It depends on the funding situation. It may well be more appropriate, once we get to a slightly later stage to then look to outside funding.
Another line of promising research that Mason thinks is unique is to look at the transfer of pigment from the pigment cells or melanocytes to the keratinocytes. She thinks this is another area where it will be possible to show that certain hormones that affect pigmentary activity in melanocytes may also either increase the donation of pigment or possibly decrease the donation of pigment. Both of those effects are important in determining the overall response.
Pigment cells are very difficult to grow in the laboratory and there are only two laboratories in Australia who are currently growing pigment cells, which require a long lead time and very much attention to sterility. They require certain chemicals to give them a boost because they donŐt easily grow. Pigment cells are not more prone to infection than others cell types but more care has too be taken against infection. This is because unlike other cells lines, which are simply thrown away if infected, skin samples are fairly rare and have to be grown for 2 to 3 months before you can get enough cells from one skin sample to actually begin experiments. Under these conditions you canŐt afford to have any infections because you will have lost several months work if that happens. The other problem is that the treatments given to skin cells in some other laboratories, to make them multiply interfere with the types of experiments that she is doing.
The work of a PhD student, Marie Ranson (who has now completed her PhD), has helped to advance the research into melanocytes by allowing them to be maintained in a situation similar to that which occurs in normal skin. That is, a situation where they don't divide a great deal but they have quite high levels of pigmentary activity. In this situation you can then experiment with treatments which might be expected to have an effect on the pigmentary function with some expectation of demonstrating whether or not they actually do have an effect.
Melanocytes in human skin normally sit on a thing called a basement membrane which separates the epidermis, the top part of the skin, from the dermis. Basement membrane is normally made by the local cells of the skin but for experiments you can get something fairly similar made from the corneas (eye parts) of cows that have been taken to the abattoirs. These cells from cow's eyes lay down a membrane that is very similar to the skin basement membrane in human skin. When melanocytes are cultured on that sort of surface they will stay there quite happily for over a week without the need for a lot of chemicals to make them grow. This is long enough to do the experiments.
If you are going to try and mimic the human skin situation as closely as possible then you should provide the cells with some sort of local basement membrane when you are growing the culture, says Mason. No other laboratory has been able to get this sort of system to go quite so well. The details of this method have already been published and it is surprising that researchers in other laboratories have not begun to copy them.
One of the interesting side lines to this research is the discovery that melanoma cells are not particularly affected by the presence of the basement membrane. They produce whatever it is that is present in the basement membrane themselves. This independence may partly underlie why melanoma cells are able to establish themselves at sites distant from the skin. They are able to travel in the body and quite happily set up shop in the liver and in other places without needing this basement membrane.
Previously in this chapter: