CELLULAR IMMUNOLOGY 82, 98-101 (1983)

Some Things That Happened on Lew Thomas’ Floor

HOWARD GREEN*

Department of Physiology and Biophysics. llaward Medical School. Boston, Massachusetts 021 I5

Received December 18. 1982

In 1957 I came to NYU Medical School, where I remained for about 13 years. I had worked there briefly a few years earlier, but this time Lew Thomas gave me a regular appointment as assistant professor in the Pathology Department. I had a nice laboratory on the First Avenue side of the fifth floor of the Medical Science Building, from which I had a view of some impressive excavations for later buildings.

In that laboratory 1 worked on many things, but mainly on cultured mammalian cells. Together with Burton Goldberg, we showed that cultured fibroblasts made collagen, and studied the regulation of its synthesis. Thereafter, cultured cells were employed for most studies on collagen biosynthesis and Burton, but not I, went on to make important contributions to that field.

Later, we began to work on somatic cell hybridization and when Mary Weiss came to the laboratory as a postdoctoral fellow, we discovered a special property of hybrids of human and mouse cells: they usually lost human chromosomes selectively, until a small number or only one remained. The selective elimination made it possible to make chromosomal assignments for remaining human genes expressed in the hybrid cells. This has continued to be the most commonly employed method for making such assignments.

On the occasion of this symposiaI tribute to Lew Thomas, 1 would like to trace the history of another line of work that began on his floor. The motivation for this work was not related to what happened later; but the connection could be understood in retrospect, and may be worth relating here.

In the 1950s Dulbecco and Vogt published their important papers on transformation of cultured mammalian Iibroblasts by polyoma virus, and I began work on this subject. I had no trouble in reproducing the essentials of their experiments but I was troubled by the difficulties of using primary or secondary cultures as the target cells for the virus. The cultures were heterogeneous and the frequency of transformation was variable. I felt that one required an established line of uniform properties, a line susceptible to the virus and whose viral transformants could be easily scored. But at that time, it was thought that established lines developed only rarely and by accident.

l This paper was presented at the Symposium, “Infection, Immunity and the Language of Cells: A Meeting in Honor of Lewis Thomas,” held at New York University Medical Center, on November 22, 1982.

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Most established lines known were not suitable targets for transformation by oncogenic viruses (the BHK line established by Stoker and MacPherson was an exception).

At that time there came along a young medical student whose name was George Todaro. He asked to spend a term working in my laboratory. He could do this because of arrangements worked out by Lew Thomas to encourage the development of sci- entifically inclined medical students. George’s work in the laboratory was extended by a summer and several more terms while he was still a medical student, and later by addition of a postdoctoral stay of several years.

When George began work, I asked him to start cultures of mouse embryo cells with a view to establishing a suitable cell line for viral transformation. In order that the process might be made reproducible, I thought that during repeated subcultivation, we should keep constant the inoculation density and transfer interval, because those two variables were likely to affect the selection pressure exerted on the cultured cells. After following this procedure we were pleasantly surprised to find that (1) we were able to establish lines routinely, (2) the properties of the lines were very sharply affected by the inoculation density and transfer interval, and (3) one could reproducibly obtain lines with the same properties by following the same protocol (later work of Aaronson and Todaro).

We called the most interesting line that we evolved 3T3 (inoculum, 300,000 cells per petri dish; transfer interval, 3 days). This line had the novel property of growing vigorously as long as the cells were sparse and stopping growth sharply when the culture became confluent (this was called contact inhibition in those days). The resting population so obtained could be stably maintained. This property made the 3T3 line suited as a target for viral transformation studies: the transformants could easily be detected through release of this growth inhibition and the formation of dense, thick colonies.

Very soon after we established the 3T3 line and began to study the effect of oncogenic viruses I noticed, in resting uninfected cultures, an occasional cluster of cells that had accumulated refractile material, sometimes so abundantly as to fill much of the cell interior. When the culture was transferred, cells containing this material could no longer be seen but they would appear again when the culture attained a resting state. This material was examined in the electron microscope by Burton Goldberg, who concluded that it consisted of lipid and not some contaminating microorganism as I had feared, I did not know how much significance to attach to this lipid, since it was accepted dogma among cell culturists that cells might accumulate lipid if they became “unhealthy.” I did think the subject might be worth investigating by someone whose main interest was in lipid metabolism and mentioned it to several such people, without any effect. I did nothing further on this subject during my remaining years at NYU, either on Lew Thomas’ floor or after I moved to the Department of Cell Biology on the sixth floor.

Some years after I moved to MIT, and in the course of other studies of 3T3 cells, I noticed an increase in the abundance of lipid-containing cells and my curiosity about their meaning was again awakened. From confluent cultures we isolated a few regions containing lipid-laden cells and cloned several lines from these isolates. This was possible even though heavily lipid-laden cells could not multiply, because the lipid began to accumulate only after the cells reached a resting state. This meant that in a well-isolated large colony, the central region might contain resting cells that had

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accumulated much lipid, while the cells at the periphery of the colony would still be free of lipid and capable of indefinite propagation. When such a clone was isolated and expanded, the cells could not be distinguished from the original culture of 3T3 as long as they remained growing, but on becoming confluent they accumulated lipid on a very impressive scale.

Over a subsequent period of several years, during which we and others studied the enzymes and hormonal sensitivity of these 3T3 clones, it became clear that the lipid accumulation was really the result of a process of differentiation into adipose cells, the same process by which adipose cells are created during embryogenesis and later. We called the process adipose conversion. Preadipose 3T3 cells had the phenotype of fibroblasts and made collagen, predominantly of types 1 and 3, but they also possessed a secondary program which, when carried out, resulted in their conversion to adipose cells. The susceptibility to adipose conversion (either high or low) was quasi-stably transmitted in different clones and therefore a heritable property of pre- adipose cells.

The adipose conversion was found to produce very extensive revision of the protein composition of the cell. Lipogenic and lipolytic enzymes appeared, together with numerous other accessory proteins and enzymes necessary to the function of an adipose cell. These changes were brought about at the level of mRNA content, since translation of cell mRNA gave a spectrum and relative abundance of proteins that faithfully reflected the biosynthetic behavior of the cells from which the mRNA was obtained.

In order that preadipose cells initiate the process of adipose conversion, two con- ditions had to be satisfied. The first was that the cells must reach a resting condition. The second was that there must be present in the medium what we called an adipogenic factor. The existence of such a factor was evident from the observation that sera obtained from different species and from animals of different ages had very different capacity to promote adipose conversion. As our attempts to isolate an adipogenic factor from serum were unsuccessful, we made extracts of many organs and assayed them for ability to support adipose conversion. Of all the organs we tested, the pituitary alone contained adipogenic activity. The activity was later found to be a property of growth hormone, but not of any other pituitary polypeptide. Many different types of growth hormone have been tested, and it now seems clear that the ability of the hormone to promote the adipose conversion of 3T3 cells can be used as an assay with good specificity and sensitivity.

The conventional view of the action of growth hormone was elaborated many decades ago and the assays established at that time are still in use today. The effect of growth hormone on the adipose conversion of 3T3 cells raises serious questions about the concept of the hormone as a simple promoter of cell growth. In preadipose 3T3 cultures cell multiplication is not affected by growth hormone. On the other hand, the number of cells differentiating into adipose cells is affected by growth hormone. The action of the hormone is direct since no other cell type is present in the system: so it does not require intermediate somatomedin production by a remote cell type.

The action of growth hormone in the 3T3 system could be described as promoting the formation of a differentiated mesenchymal cell type. It should therefore be asked whether the hormone might not also promote the differentiation of other mesenchymal

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cell types from their precursors. For example, it is possible that growth hormone can increase growth of the skeleton by promoting the formation of chondrocytes from prechondrocytes?

In retrospect, it is possible to guess why the 3T3 line possesses the ability to undergo adipose conversion. Cultures made from disaggregated cells of late mouse fetuses are likely to contain preadipose cells, since the adipose tissues of the mouse develop early after birth. Under ordinary conditions of cultivation, in which the cells are not kept continuously in exponential growth, preadipose cells would likely convert to adipose cells. This can be commonly seen in primary or secondary cultures allowed to become dense. Since maturing adipose cells lose the ability to multiply, any preadipose cells would, under such conditions, tend to be eliminated from the population. It seems likely that the same culture conditions that made the 3T3 line valuable as a target for viral oncogenesis also made it preserve the program for adipose conversion.

Considering the matter more generally, we may ask, what really happened on Lew Thomas’ floor? I think it was the creating of possibilities and initiatives. His floor was a place fertile in ideas and intuitions, and these gave rise to new directions in research, some of which are still current today.

It is impossible to state precisely which of Lew’s influences brought this about, but I think two were most important. First his enthusiasm, which not only raised everyone’s spirits, but also dissolved existing frameworks and made possible the development of new ones. The second was his generosity in establishing and setting free young scientists. Generous treatment, once received, is likely to be returned to others. So I believe that what Lew Thomas gave to members of the Pathology Department is even now being regiven, both there and elsewhere in the world.