Hoofdstuk 133

17 maart 2004

 
Background information on my ten most quoted articles
or
John Hilkens, Arnout Sonnenberg made the highest scores
and
Links with the work of the Nobel Laureates Milstein, Dulbecco, Temin and Varmus

Dear Peter,

My most quoted article is from the year 1984 and the first author was John Hilkens (See below, Pubmed Nr 6.206.003 or the six-million-two-hundred-six-thousand-two-hundred-third paper in the system). I have worked with him in my laboratory since around 1975 - when I offered him a position on my American grant at the time - until I left the AvL in 1988 for the VU.

I had sent him to Frank Ruddle in Yale to learn somatic cell genetics and when he came back directed him to generate the first hybridomas in order to create so-called monoclonal antibodies, which was started in april 1977. It proved to be the most important decision in my scientific life as a researcher doing fundamental work.

And my group was the first to start this new technology in Holland receiving the myeloma cells from Cesar Milstein upon my request exactly on April ten 1977 - described in August 1975 in Nature and for which Kohler and Milstein, working together in Cambridge, England, got the Nobel Price - although colleague Dr. Claus Vennegoor of the Immunology Department of Philip Rumke at the time was the first one to identify an antigen recognized by monoclonals. It was actually a mycoplasm infecting the melanoma cells with which she had immunized the mice. The second laboratory which started in Holland still in the seventies was that of Organon and they created antibodies against human chorionic gonadotropin.

I had chosen milk fat globule membranes from human milk as our antigen and eventually after much trial and error we created a series of almost twenty monoclonal antibodies against the various molecules of these membrane, one of which, at the time called MAM-6 (now first mucin or MUC-1), proved to be a major component of the mammary cancer cell membrane as well. Our work which was started in the spring of 1977, yielded as its most important paper the one mentioned below on top of the list in 1984 with John Hilkens as principal author.

John was my closest associate at the Netherlands Cancer Institute, who already got his permanent position early in the eighties. He is still active in the MUC1 field to this day, but the mucin field has never caught on as a major topic in the Institute. Even John has moved into other areas of research. I myself, when I left for the Vrije Universiteit continued to study the natural immune responses against this molecule and its expression and function in the human body. One of my theories has always been that this huge slimy molecule is a protective shield on the carcinoma cells in the human body, protecting the cells from immune attack amongst other things.

The paper in the International Journal of Cancer had 397 quotations or in other words its SCI is 397, whereby SCI stands for Science Citation Index, since 1984 and until the end of 2003, which is 20 years so it has collected an average of 20 citations per year. Such a highly quoted piece of work can be called a "classic". In my case it is the best classic and we have become well known because of it. People in our field called themselves The Mucineers in the nineties and still.

John got his doctorate with Prof. Dr. Piet Borst and me as co-promotor in the mid eighties, shortly after Piet got his position as scientific director at the AvL, the Antoni van Leuwenhoekhuis.

The second most quoted article came as a total surprise and it shows how science can lead you in completely unexpected directions. It was the work of Arnout Sonnenberg funded by a KWF (Queen Wilhelmina Fund) project which I obtained in the early eighties. Arnout had been sent by me to study in the USA with the Nobel Laureate Renato Dulbecco at the Salk Institute in San Diego and he came back with completely new ideas.

Dulbecco got his price for being able to grow cells from animals in tissue culture and to develop media with the appropriate food stuffs in it to keep cells alive in so-called T flasks under sterile conditions of course. And at the time Arnout was in the States Dulbecco's interest was in growing stem cells of the mammary gland and try to make them differentiate in vitro, in these flasks. In order to measure differentiation he used antibodies to various molecules appearing and disappearing through the various stages from a stem or embryonic cell to a mature milk producing cell. He worked with rats and used monoclonal antibodies made in the mouse.

Back home Arnout found out that we never worked with rats as a model, but with mice and because he wanted to do similar work as his teacher, he started to develop cell lines from mouse mammary tumors. But in order to test for molecules shifting in their appearance on the cells under different conditions of growth and with different substances added to the medium, it was for technical reasons difficult to work with mouse monoclonal antibodies.

At the time some group elsewhere in the world had succeeded for the first time to generate rat monoclonal antibodies, by no means an easy feat. But Arnout went to Belgium to get this special inbred strain of rats for his purpose and lo and behold he created a fantastic set of rat antibodies to a mouse mammary cancer. He changed the practice of naming the antibodies and used the names of all the girls of various laboratories to be included in the antibody name. One of the antibodies was for example called Gonnie-H3 or GoH3.

This all happened in the early eighties and one of his papers inspired by the great Dulbecco is now number 4 on the list. For this paper he used an antibody from his rat series, which later - after his cum laude thesis with Piet Borst and me - when he had moved to the laboratories next door of the Netherlands Blood Transfusion Services proved to be reactive with human blood platelets. Totally and completely unexpected. Even looking for such a thing was kind of strange and weird experiment, but Arnout apparently did and later found out that this antibody identified a complex of two antigens on the human platelet which is called complex Ic/IIa. It changed his career.

The finding was published in the prestigious Journal of Biological Chemistry and proved to be of utmost importance for all people in this world working with human platelets which as you know have a function in blood clotting. There were no antibodies to this complex at the time and the rat antibody actually reacted with the active site of the complex and could block the function of the protein complex. So the paper became highly quoted and now has 320 quotations since 1987 and say 16 years by now. So the average number of quotations per year is around 20 per year, actually quite similar as this number for paper number One from Hilkens.

Arnout has later moved back to the Netherlands Cancer Institute again because of his excellence and scores very highly in number of papers per year at almost one per month throughout the nineties and until now. He has become a top scientist in the world of adhesion molecules, molecules which keep cells and tissues together, one group of which is called the integrins, which he co-discovered at the time using panels of monoclonal antibodies. And also molecular probes to look at the genes for these adhesion molecules. Various familiar skin diseases are due to mutations in such genes for adhesion of cells.

This direction was greatly influenced by the fact that he left the NKI and worked for quite a while in the CLB as we call it, under Von dem Borne. He had to stop with the research on stem cells which he picked up at the Salk Institute and he never went back to it. This was in retrospect a loss and as a consequence the NKI does not have a strong tradition in stem cell research, which has become so important in this day and age. The molecular biological direction of Piet Borst, the Director, prevailed above my direction as a tumor biologist.

Whereas John Hilkens maintained his traditions and stayed with the mucins, Arnout - by accident and virtue of antibody specificities - left the field of tumor and stem cell differentiation in order to study the molecular mechanims of cell attachment.

Paper Number 4 of Sonnenberg - published in one of the best pathology journals called Journal of Histochemistry and Cytochemistry - got sofar 140 quotations since 1986, an average of about 8 citations per year, still quite a classical paper which will be quoted for a long time to come if you ask me.

The third most quoted article is from the good old days when I was studying in Memorial Sloan-Kettering under Dr. Lloyd Old and with Bob Nowinski as my closest associate. I was learning immunology and developed in that Institute the so-called immunofluorescence technique to detect viral antigens of the group which was called oncogenic RNA viruses or oncornaviruses. The mouse mammary tumor virus and various leukemia viruses from chickens, mice and cows among others belonged to this group. Through these viruses the first so-called oncogenes were discovered by Varmus and Bishop who received the Nobel price for their work.

The immunofluorescence technique is now mainly used in Flow Cytometry to measure the positivity of individual cells tagged through the antigen/antibody complex with the fluorescent probes. It may be one of the most important immunological methods ever detected and I was one of the pioneers in it. But the real pioneer was Ploem, an electonmicroscopist in Leiden who was among the first to use these fluorescent dyes to attach to proteins.

In the mid seventies I sent one of my doctoral students Roeland Nusse to the laboratory of Varmus and he started to work on the so-called integration sites of int genes, which proved to be of enormous importance for carcinogensis of epithelial cells, so much that Nusse, now Howard Hughes Professor at Stanford Univesity in California, still works in this field as a famous scientist and student who started his career at the AvL.

The paper published in 1972 in the prestigious American journal Cancer Research is a classical one with 177 quotations to date so about 6 per year since it appeared over 30 years ago. Later I used the method in Howard Temin's laboratory in McArdle in Madison, Wisconsin where I worked briefly to determine that an oncogenic RNA tumor virus put on a so-called synchronized culture of cells in vitro only produces the viral antigens after one round of DNA replication.

This was amongst indirect but definitive proof that an enzyme exists in cells which transcribes RNA of the virus into its DNA template which actually gets into the host DNA or more simply the chromosomes, before another enzyme (transcriptase), makes RNA again as a prelude to new virus particles. Only at that time enough viral antigen or protein appears in the cell to be detected by my immunofluorescence method.'

Temin got the Nobel Prize for these findings together with Baltimore and Mizutani and in that year my teacher Peter Bentvelzen was on the short list for the Price as well,
because he proved that the mammary tumor virus in a particular mouse strain which we called GR strain, is inherited as a single gene, in other words must have a DNA copy somewhere in the chromosomes of that mouse strain. And it causes mammary cancer in that mouse where that disease simply is a single gene disease and the gene is dominant.

Bentvelzen's work was another kind of proof that the RNA can change into DNA and from that time on, it was proven that actually RNA is a more primitive and earlier molecule in the living world than DNA.

Much of what I tell here was actually the content of a review which Peter Bentvelzen, my closest teacher under Otto Mulhbock, which now appears at rank 7 of my list and was published in Advances in Cancer Research in 1978 with 92 citations over 26 years, an average of close to 4 per year and probably not quoted anymore for quite some time.

The remaining papers of the first ten most quoted article of my list were not the result of a major line of research in my own laboratory, but rather the consequence of international collaboration with
(1) A Belgian group with Heremans and Billiau working on interferons (Nr. 5 with 115 citations),
(2) The group of the famous George Klein, Member of the Nobel Committee in Stockholm - where I worked for some time with Beverly Reedman - on the Epstein-Barr virus (Nr. 6 with 105 citations).
(3) The group of Jan-Willem Arends of the University of Maastricht when he was professor in pathology (Nr. 8 with 90 citations),
(4) The Indonesian veterinary dokter Ressang at the Centrale Diergeneeskundige Instituut on bovine leukemia viruses (Nr. 9 with 83 citations) and finally
(5) The group of Beverly Mock at the NIH in Bethesda, USA, on lymphotoxin and tumor necrosis factor genes (Nr. 10 with 75 quotations).

These 10 most quoted articles of my 193 in Pubmed and my 330 papers in total score a total of close to 1500 citations of about 4700 as grand total, so roughly one third of all citations. And I now realize that this is a good way to write your "real" scientific CV or resume. But the prize for drawing the SCI is around 1000 Euro, so it is not the cheapest way to write your CV based on scientific content.

Dear Peter,

This gives you a view, a glimpse, of my work as a young researcher in the seventies and eighties, dedicated to fundamental cancer research, working in a great Institute, the second oldest Cancer Institute of the world - named after the most famous Dutch researcher in the world Antoni van Leeuwenhoek, the discoverer of the microscope - which was founded in 1914 on March the 30th and for which the 90th birthday will be held on that same day a few weeks from now.

And it shows you that I was forever seeking international collaboration and good teaching for my closest students, some of which were sent to Nobel Laureates to study and come back with new methods and new ideas.

So my inevitable first slogan now is: Boost your SCI through your most intelligent students.
And from it follows the second slogan: Boost your SCI through international collaboration with top scientists.

Not only as a scientist in Holland or for that matter anywhere in the rich world, but more so in a country like Indonesia.

So the same I am trying to do again - now at the end of my career - already for as much as six years, since 1998 here in Indonesia and so far so good. Two biologists from Gadjah Mada - Dewayani Purnomosari and Jajah Fachiroh - are doing their PhD work in Holland with Prof. Dr. Paul van Diest and Prof. Dr. Jaap Middeldorp at the VU, while Savira Ekawardhani from ITB and Sanbe has found on her own strength a place in the laboratory in Trier with Prof. Dr. Jobst Meijer and Yunia Sribudiani has just returned from a stay in Germany with Prof. Dr. med. Magnus von Knebel Doeberitz in Heidelberg.

With warm personal regards,

Doctor Jo the not-so-modest scientist

1               397             HILKENS J 1984
{135} Hilkens J, Buijs F, Hilgers J, Hageman P, Calafat J, Sonnenberg A, van der Valk M.  Monoclonal antibodies against human milk-fat globule membranes detecting differentiation antigens of the mammary gland and its tumors. Int J Cancer 1984; 34 (2): 197-206.
PM:6206003

2               320             SONNENBERG A 1987
{92} Sonnenberg A, Janssen H, Hogervorst F, Calafat J, Hilgers J.  A complex of platelet glycoproteins Ic and IIa identified by a rat monoclonal antibody. J Biol Chem 1987; 262 (21): 10376-10383.
PM:3301835
                        
3               177             HILGERS J 1972
{221} Hilgers J, Nowinski RC, Geering G, Hardy W.  Detection of avian and mammalian oncogenic RNA viruses (oncornaviruses) by immunofluorescence. Cancer Res 1972; 32 (1): 98-106.
PM:4332476

4               140             SONNENBERG A 1986
{103} Sonnenberg A, Daams H, Van der Valk MA, Hilkens J, Hilgers J.  Development of mouse mammary gland: identification of stages in differentiation of luminal and myoepithelial cells using monoclonal antibodies and polyvalent antiserum against keratin. J Histochem Cytochem 1986; 34 (8): 1037-1046.
PM:2426332

5               115             HEREMANS H 1978
{181} Heremans H, Billiau A, Colombatti A, Hilgers J, de Somer P.  Interferon treatment of NZB mice: accelerated progression of autoimmune disease. Infect Immun 1978; 21 (3): 925-930.
PM:213392

6               105             REEDMAN B M 1974
{214} Reedman BM, Klein G, Pope JH, Walters MK, Hilgers J, Singh S, Johansson B.  Epstein-Barr virus-associated complement-fixing and nuclear antigens in Burkitt lymphoma biopsies. Int J Cancer 1974; 13 (6): 755-763.
PM:4136722

7               92              HILGERS J
{193} Hilgers J, Bentvelzen P.  Interaction between viral and genetic factors in murine mammary cancer. Adv Cancer Res 1978; 26 143-195.
PM:204164

8               90              ARENDS J W
{149} Arends JW, Verstynen C, Bosman FT, Hilgers J, Steplewski Z.  Distribution of monoclonal antibody-defined monosialoganglioside in normal and cancerous human tissues: an immunoperoxidase study. Hybridoma 1983; 2 (2): 219-229.
PM:6381289

9               83              RESSANG A A 1974
{213} Ressang AA, Mastenbroek N, Quak J, van Griensven LJ, Calafat J, Hilgers J, Hageman PC, Souissi T, Swen S.  Studies on bovine leukemia. I. Establishment of type C virus producing cell lines. Zentralbl Veterinarmed B 1974; 21 (8): 602-617.
PM:4372838

10              75              GARDNER S M 1987
{93} Gardner SM, Mock BA, Hilgers J, Huppi KE, Roeder WD.  Mouse lymphotoxin and tumor necrosis factor: structural analysis of the cloned genes, physical linkage, and chromosomal position. J Immunol 1987; 139 (2): 476-483.
PM:2885372