The Showe laboratory is studying patterns of gene expression in healthy and diseased tissues using customized microarrays of complementary DNA (cDNA). To create the microarrays, probe cDNA representing thousands of genes of interest is spotted onto a nylon filter in a grid. Target RNA is then extracted from the tissues to be investigated, and copies of the RNA are produced that incorporate a fluorescent or radioactive tag. When brought into contact with the DNA probes on the microarray, the RNA targets bind to the probes corresponding to the genes that generated them. The resulting pattern of DNA-RNA complexes are read with a scanner that detects radioactivity or flouresence and quantified by computer to assess not only which genes are active in the study tissues, but also how strongly they are expressed.
Using microarrays, researchers can evaluate and compare the relative expression in different tissues of an extensive set of genes. For example, using cDNA microarrays with probes for 5,000 genes, the Showe team analyzed gene expression patterns in samples from 49 patients with Sezary syndrome - an aggressive form of cutaneous T-cell lymphoma (CTCL) - and 22 healthy normal controls. Results identified a number of new genes whose changes in expression may contribute significantly to the development and progression of CTCL. Expression patterns of as few as four genes were shown to be reliable for classifying patients with very low tumor burdens, and expression of a single gene was able to identify Sezary Syndrome in 80 percent of the patient samples analyzed.
Also, researchers in the Showe laboratory have been using a mouse model to study gene expression patterns associated with the development of septic shock. Mice have no adverse effects from being treated with small non-toxic doses of the bacterial byproduct lipopolysaccharide (LPS), but if they are infected with an attenuated strain of mycobacterium three weeks before low-dose LPS is administered, an inflammatory response is induced that results in septic shock and death. The research team found that low-dose LPS causes significant changes in gene expression although mice remain healthy.
The research team is using cDNA microarrays to characterize gene expression patterns in a human monocytic cell line after infection with Chlamydia pneumoniae, which has been implicated in atherosclerosis and also CTCL. The array generated for these studies carried probes for 2200 genes known to be associated with inflammation. The studies show a significant alteration in gene expression in infected cells, including cell surface markers, cytokines, and chemokines and their receptors, as well as genes associated with thrombosis and tissue remodeling whose expression could contribute to atherosclerosis. The laboratory has also begun collaborations to study differential gene expression in hibernating animals throughout the year as a model of obesity as well as gene expression in blood samples from multiple sclerosis patients that correlate with responsiveness and non-responsiveness to therapy.
Recent Scientific Advances
Gene Expression in Cutaneous T-Cell Lymphoma
As part of the National Cancer Institute's Directors Challenge, a program to develop molecular techniques for the diagnosis of cancers, the Showe laboratory has been using cDNA microarrays to study gene expression patterns in samples from patients with cutaneous T-Cell lymphoma (CTCL), an indolent cancer that is incurable except in the early stages. This work is directed at identifying patterns of gene expression specific to CTCL that could provide better criteria for diagnosis, prognosis, and response to therapy. There are two major forms of CTCL. Mycosis fungoides (MF) is skin associated, progresses slowly, and is difficult to differentiate from other benign skin diseases in its early stages when it is most treatable. Sezary syndrome (SS) is the more aggressive leukemic form of CTCL, with circulating malignant cells and is resistant to therapy.
The work on CTCL is being carried out with clinical collaborators at the University of Pennsylvania (Dr. Alain Rook, Dermatology) and Johns Hopkins University (Dr. Eric Vonderheid, Dermatology.). Initial studies have focused on patients with Sezary syndrome. Using cDNA microarrays carrying probes for 5000 genes, the investigators have analyzed gene expression patterns in samples from 49 patients and 22 healthy normal controls. Array results have confirmed many previous observations and have also identified a number of new genes whose changes in expression may contribute to the development or the progression of CTCL. Expression patterns of as few as four genes can be used to classify patients with very low tumor burdens and expression of a single gene, not normally expressed in lymphocytes can be used to identify SS in 80% of the patient samples analyzed. They have also been able to identify gene expression patterns that identify patients with very poor prognosis even when they have very low tumor burdens.
This is one of the first attempts to apply this technology to patient samples with low tumor burdens. The collaborators are currently analyzing additional CTCL patient samples and increasing the numbers of genes assayed in order to confirm and extend results. Analysis of additional patients is being carried out to determine whether the patterns of gene expression they detected in SS patients are also associated with MF. The goals of these studies include the identification of a small number of genes that could easily identify early MF patients, identify gene expression patterns associated with poor prognosis, and then discover new targets for therapy.
The development of sepsis is responsible for large numbers of deaths associated with serious injury. The research team has been using a mouse model to study complex gene expression patterns associated with the development of shock. It has been shown that mice have no adverse effects from being treated with small non-toxic doses of the bacterial product lipopolysaccharide (LPS), but if they are infected with an attenuated strain of mycobacterium (BCG) three weeks before low dose LPS is administered, an inflammatory response is induced that eventually results in septic shock and death. It has been shown that inactivation of interleukin 12 (IL-12) - a cytokine that is critically important for the inflammatory response - with an antibody against IL-12, protects the treated mice from developing sepsis. Although these results suggested IL-12 expression was central to the septic response, it was found that IL-12 levels are similar in the LPS and BCG/LPS models. In trying to understand these observations, the team examined the levels of message that are induced for the IL-12 receptor (IL-12R) genes under both conditions in mouse spleen. They found that the expression of the IL-12R beta2 gene that is required for IL-12 signaling is not induced by LPS alone and message levels for cytokines such as IFN-gamma remain low. Unlike the mice with LPS alone, BCG-primed mice respond to LPS with a rapid induction of the message for the beta 2 chain of the IL-12R. This suggested BCG infection primed the expression of IL-12 receptor genes.
In general cDNA microarrays help to determine the effects on global gene expression in this system. The laboratory found that low dose LPS causes significant changes in gene expression although the mice remain healthy. Injecting the mice with low dose LPS after priming with BCG also causes many changes in gene expression, but in many cases the effect on gene expression is the reverse of what is found with LPS alone. Understanding the more global effects on gene expression associated with sepsis may lead to new ideas on how to treat it.
The Role of Chlamydia pneumoniae Infection in Chronic Disease
The association of pathogenic infections with chronic diseases has come under greater scrutiny since the identification of the role of Heliobacter pylorii in the development of ulcers and stomach cancer. Accordingly, a recent study addressing this question has suggested that inflammation may be a better predictor of heart failure than cholesterol levels. The research team has been studying whether infection with Chlamydia pneumoniae may play a role both in the development of atherosclerotic plaques and in the development or progression of CTCL. Since C. pneumoniae can infect monocytes/macrophages and T-cells, it is possible that the monocyte population associated with an atherosclerotic plaque could either become infected at the site of inflammation or may be infected before recruitment and carry the infection to the site of inflammation.
To test this possibility, the team used cDNA microarrays to characterize gene expression patterns in the human monocytic cell line after infection with C. pneumoniae. The array generated for these studies carried probes for 2200 genes known to be associated with inflammation and included genes coding for cytokines and chemokines, programmed cell death proteins, cell cycle enzymes, cell surface markers, and tissue-specific homing receptors, as well as genes associated with clotting and thrombosis. The studies show a significant alteration in gene expression in infected cells including cell surface markers, cytokines, and chemokines and their receptors, as well as genes associated with thrombosis and tissue remodeling whose expression could contribute to atherosclerosis.
The results from these studies are now being confirmed in primary human monocytes and in a mouse model for the development of atherosclerosis. For these studies the investigators are developing methods for isolating RNA from the small numbers of cells associated with atherosclerotic plaques that develop in these animals. This work was initiated in collaboration with Dr. Eva Gonczol, presently at the Johan Bela National Center of Epidemiology, Budapest, Hungary. The number of genes that will be surveyed in the new studies will include almost 20,000 human clones with a similar number available for the mouse studies.
Gene expression profiling in a model for obesity
The Showe laboratory is also collaborating with Dr. Bert Boyer, University of Alaska at Fairbanks, on studies of hibernation in the Arctic ground squirrel as a model for obesity. Since cDNA clones are not available for ground squirrels, the researchers are using the mouse 9600 gene arrays developed by Showe and colleagues. Depending on cross-hybridization between homologous genes in the two species, the investigators aim to detect gene expression changes during the transition between euthermia and torpor, the state of deep hiberbation. A pilot study undertaken to determine the feasibility of this approach was very promising - the percentage of mouse genes recognized by ground squirrel messenger RNA was comparable to that responding to mouse RNA. A more complete study of additional animals is presently underway.
Gene expression in multiple sclerosis
In a recently initiated collaboration with Dr. Cris Constantenescu, Nottingham, U.K., the Showe laboratory is examining gene expression profiles in samples from multiple sclerosis patients involved in clinical trials with immuno-modulatory drugs. The aim is to determine whether it is possible to identify gene expression patterns in blood samples that correlate with responsiveness and non-responsiveness to therapy. If successful, these studies could lead to more informed choices for therapy.