50 Years of Basic Research – A Personal History

 

 

50 Years of Basic Research – A Personal History: David T. Denhardt

As published in the  2010 issue of COOT, the Newsletter for the Class of 1960, Swarthmore College

In my senior year at Swarthmore I faced two momentous questions: 1) Could I persuade Georgetta Harrar to marry me, and 2) knowing the answer, where to go to graduate school? She finally said yes, so I chose the largely male CalTech over CoEd Berkeley. Given CalTech, what would I do there? About that time I came across an article in the NYT described exciting research by a new member of the CalTech faculty (Robert Sinsheimer) on a bacterial virus (phage ФX174) that had single-stranded DNA as its genome. This was unprecedented (all DNA was then considered to be double-stranded) and I decided that I wanted to work on that virus. It was my good luck to have spent the previous summer doing research at CalTech (with Ray Owen, a renowned immunologist) so I had some connections; Ray, a wonderful person, made possible my first two scientific publications (in 1960, in the field of immunology).

After graduation, Getta went to Eugene, OR to work with George Streisinger, previously of Cold Spring Harbor where we both met him, a well-known “phage person”. (She wanted a year of independence before tying herself down.) Fortunately, Eugene was “only” a 14-hour bus ride north of Pasadena. That first year I roomed with John Urey (who recently convinced me that someone might actually be interested in reading this “memoir”). A second Swarthmore classmate, Ellen Glowacki (Strauss) also came to CalTech to pursue her graduate work. That first year (1960-61) the three of us struggled to make our way, learning to enjoy Almaden wine and living through the Cuban missile crisis (among various other crises).

Getta and I married in June 1961 and we moved into a one-room apartment in Pasadena, one block from CalTech. The remaining three years at CalTech I focused on elucidating aspects of ФX174 replication while Getta joined the Edgar-Delbrück group to perform some seminal studies on the biology of phage T4. (She also enjoyed many evenings of bridge with other members of the Sinsheimer group, including Jim Strauss, who later married Ellen.) In 1963 we moved to a larger apartment in Pasadena to accommodate our first child, Laura Jean. In 1964 the CalTech Biology department granted me a PhD in Biophysics, thanks in part to the willingness of the Physics department to realize that I would never really understand what a Fourier equation was. My PhD thesis on the replication of ФX174 single-stranded DNA was published in four papers in the J Molecular Biology in 1965.

In the summer of 1965 we drove east to Cambridge, MA, where I had accepted an Instructor position in the Department of Biology to teach, along with several others, an undergraduate biology laboratory in a course for non-scientists taught by George Wald. I also set up my laboratory to pursue the goal of achieving in vitro DNA replication using ФX174-infected E. coli as the model system. In the course of this work I published a method to detect specific DNA sequences (ФX in this case) using a DNA-DNA hybridization technique. Some years later Ed Southern immortalized this methodology in his ground-breaking Southern Blotting procedure when he christened the somewhat exotic buffer I had developed to enable his technology as “Denhardt’s buffer”. Among our contributions to knowledge during this period was the identification of a bacterial gene (the rep gene) essential for ФX replication that encoded an enzyme, a helicase, important for DNA unwinding. Having failed to demonstrate in vitro DNA replication (a long story), and as expected not receiving tenure at Harvard, I accepted an offer (by the new Chair of the Biochemistry Department, Angus Graham) to become an Associate Professor of Biochemistry at McGill University in Montreal, Quebec. Very shortly after moving there, with three young children in tow, terrorist elements in the Parti Quebecois murdered the British trade minister and kidnapped a Quebec cabinet minister. This brought Canadian Troops into the streets of Montreal and a declaration of martial law by Pierre Trudeau, the Prime Minister of Canada. During our very enjoyable (and peaceful) 10 years in Montreal we saw it become increasingly more French. (Unfortunately, my hope to become fluent in French was dashed.) In the lab I pursued various studies, mostly focused on DNA replication of ФX in E. coli, and in the later years mammalian viruses in mammalian cells. We also enjoyed a wonderful year (1978) touring parts of Europe with our three children while I was on sabbatical leave at the Imperial Cancer Research Fund Lab in London, UK, where I participated in some studies on a protein called p53 in Lionel Crawford’s group.

In 1980 the Canadian Cancer Society convinced me to become Director of the Cancer Research Laboratory at the University of Western Ontario in London, Ontario. A major attraction was good funding for at least five years to pursue whatever avenues of research I chose to pursue. There I applied the recently developed DNA cloning technology to several cancer-relevant projects, notably DNA replication and tumor promotion in mammalian systems. The outcome of these studies was the cloning and identification of a number of mouse genes whose function(s) were poorly understood. One of these genes, which was to become very important a few years later, was osteopontin. The 9 years in London marked the completion of a transition from my life studying DNA replication in unicellular prokaryotic systems to investigating diverse phenomena in mammalian cells, and subsequently in mice. One unanticipated consequence of our move to Canada in 1970 was that our children received their early education there and thus grew up, not surprisingly, considering themselves as Canadians. (This was perhaps appropriate given that an ancestor, a Hessian soldier who had fought for the British during the Revolutionary War, moved to Canada when the war was over. My grandfather moved to the US in the mid 1800s; my father was born in Utah.)

In 1988 I accepted the Chairmanship of the Department of Biological Sciences at Rutgers University in New Brunswick, NJ. Reasons for the move (besides returning home to the USA) included excellent research funding for a number of years and proximity to Getta’s mom in Scarsdale NY. (My family had moved to Britain many years earlier.) Getta was gainfully employed, first by Johnson & Johnson and then by Schering Plough, until her retirement a few years ago. A drawback of the move is that we left two “children” in Canada, Kristin and family near Toronto and David and family, now in Calgary. Our oldest daughter, Laura, had moved to Greensboro NC some years earlier.

During the first few years at Rutgers we continued our studies on three genes that we had cloned at UWO, TIMP1, MRP/proliferin, and osteopontin. By mid 1990 it became apparent that osteopontin (OPN) was the most intriguing of the three and of potentially broader significance. We had created a “knockout” mouse unable to make OPN and somewhat to our surprise this mouse appeared to be completely normal. (Typically mice engineered to lack a specific gene exhibit a phenotype different from what was anticipated.) A long-time collaborator, Masaki Noda, in Japan who had an interest in OPN asked me to send him some of the mice. An MD whose research focused on bone pathologies, he decided to see what would happen if some female mice were ovariectomized; to his surprise they did not lose bone mineral (i.e. did not undergo osteoporosis). That was the first phenotype for our OPN KO mice.

Since retiring as Chair in 1995 I have had fun (second childhood perhaps?) participating, with collaborators and students, in elucidating important functions of OPN in mammalian systems physiology. It is both a secreted protein found in all body fluids (a cytokine) and an extracellular matrix protein in mineralized ECM; there is also intracellular form of OPN whose function is obscure. Structurally, OPN is a highly charged, flexible molecule that interacts with multiple cell surface receptors. As a protein in the bone matrix, it is essential for osteoclast function; these are the cells that normally degrade bone in the normal process of bone turnover, thereby explaining the absence of osteoporosis. As a protein overproduced by almost all metastatic cancer cells, it stimulates the migration and tissue invasion of cancer cells. Importantly, we and others have shown that OPN acts on cells to help them survive under conditions where they would otherwise die. It involved in various immune responses, including the atrophy of the spleen and thymus in response to stress and the progression of autoimmune disease. Recent studies have revealed its presence in many inflammatory situations, including degenerative brain diseases. Like many cytokines, OPN’s action can be either beneficial or harmful. A current direction of our research is to ascertain whether any of the anti-OPN monoclonal antibodies we have created in the last few years may be useful in particular clinical settings, for example inhibiting the progression of auto-immune disease or cancer metastasis. For more details, please see http://lifesci.rutgers.edu/~denhardt

I have had the good fortune to be able to interact with numerous students and colleagues over the past 50 years; they are largely responsible for whatever successes we have had in furthering our understanding of biological systems. Our research in the US was generously supported by NSF and NIH, support that today would be almost impossible given the cultural changes at NIH. Last but not least, I acknowledge my great debt to Getta, whose love and support made possible much of the above.