The History of Leukemia Therapy

In the bone marrow or peripheral blood, the rare hematopoietic stem/progenitor cells become mature white blood cells after proliferation and differentiation and then continuously replenish the dead blood cells. If the proliferation and differentiation process is dysregulated, the hematopoietic stem/progenitor cells remain in various immature stages and their number continue to increase, which leads to the development of leukemia. Thus, leukemia disorders are caused by the abnormal control of the proliferation and differentiation of the hematopoietic cells. Scientists have been looking for cytokines or drugs which can promote the differentiation of leukemia cells while inhibit their proliferation in order to cure leukemia [1].

In the 1980s, a variety of cytokines, which regulate the proliferation and differentiation of hematopoietic cells, such as G-CSF (granulocyte colony stimulating factor), M-CSF (macrophage colony stimulating factor), GM-CSF (granulocyte macrophage colony stimulating factor), Multi-CSF (now known as IL-3) and IFN-γ, were discovered. The common feature of these cytokines is that they do not only promote the differentiation of hematopoietic cells but also stimulate their proliferation. The use of these cytokines for leukemia therapy poses a problem in that although they will lead to differentiation of the leukemia cells the number of the leukemia cells will simultaneously increase significantly, which may worsen rather than cure the leukemia. Therefore, scientists were still looking for a cytokine, which only induces the differentiation of leukemia cells without promoting their proliferation in order to find a cure to leukemia.

From D-Factor to LIF

In 1969, during the establishment of a cell line (named M1, i.e., the well-known M1 myeloid leukemia cell line, which is commonly used in the evaluation of the biological activity of recombinant mouse LIF) from a spontaneous myeloid leukemia of SL strain mice, it was found that the cell line could be differentiated into macrophages or neutrophil granulocytes when cultured with a conditioned medium from normal cells [2]. The unknown active components in the conditioned medium from normal cells were named “Differentiation stimulating factors”, and referred to as D-factors.

In 1981 [3] and 1984 [4], MGI-2 (Macrophage and

Granulocyte Inducer type-2) and D-factor were identified from the culture media of mouse Krebs sarcoma cells and mouse L929 fibroblast cells, respectively, both of which can induce the differentiation of M1 myeloid leukemic cells but not stimulate the proliferation of normal hematopoietic cells.

Subsequently, in 1987 Donald Metcalf’s lab in Walter and Eliza Hall Institute (WEHI), Royal Melbourne Hospital in Australia identified the protein which could induce the differentiation of M1 myeloid leukemia cells from the culture medium of mouse Krebs sarcoma cells. This protein was named leukemia inhibitory factor, i.e., LIF because it can inhibit the proliferation of M1 myeloid leukemia cells. Meanwhile, it was found that this protein could not stimulate the proliferation of normal myeloid precursor cells. Based on the fact that all of these factors are able to induce the differentiation of M1 myeloid leukemia cells into macrophages, Daonald Metcalf et al. assumed that LIF and the previously found MGI-2 and D-factor are actually the same factor. Since then, LIF became the formal name for this factor, while both MIG-2 and D-factor are aliases of LIF. Later on, the genes of mouse LIF and human LIF were cloned by the lab of Donald Metcalf in 1987 and 1988, respectively [5,6].

The discovery of LIF appeared to bring hope for an effective treatment for leukemia. However, in-depth studies found that LIF has a wide array of activities.  Even for leukemia cell lines, LIF can be either the inducer or inhibitor of their differentiation. Thus, similar to other pleiotropic factors such as TNF-α, the path of LIF to clinical applications is not simple. Over the years, LIF has attracted considerable attention from hematologist, neurobiologists, muscle cell biologists, bone biologist, endocrinologists etc., all involved in the study of LIF.

LIF is thus characterized as a pleiotropic cytokine with effects on various cell types and organs [7], yet its mechanism of action is not fully elucidated.

LIF Roles in mESC Culture

LIF got its name due to its capacity to inhibit the proliferation of M1 myeloid leukemia cells. Then, how did the scientists find its crucial role in the culture of mESC? The scientific research is full of major discoveries that were made by coincidence, thus it needs a keen and original thinking in order to identify the significance of these coincidences.

In 1981, Martin Evans and Matthew Kaufman (Cambridge University) and Gail Martin (The Univeristy of California, San Francisco (UCSF)) obtained mESC from mouse embryonic inner cell mass (ICM). At that time, it was necessary to culture mESC on feeder cells in order to avoid spontaneous differentiation of the mESC [8,9].

In an attempt to identify the reason for the inhibition on the differentiation of mESC by the feeder cells, a few inhibitory compounds were separated from the culture supernatant of feeder cells [10, 11]. However, all these compounds showed only weaker inhibition of mESC differentiation compared to the DIA (Differentiation- Inhibiting Activity) which was isolated from the culture supernatant of Buffalo rat liver cells by Austin Smith (University of Edinburgh Medical School) in 1986 [12]. Later, Austin Smith moved to the Oxford University where he continued his research on DIA. In 1998, in cooperation with the Genetics Institute (USA), Austin Smith found that DIA was capable of supporting continued growth of DA-1a MoMuLV-induced leukemia cells when investigating the possible actions of DIA in non-ES cell systems [13]. Coincidentally, the Genetics Institute has isolated a complementary DNA clone (pC10-6R) encoding a factor named human interleukin for DA cells (HILDA). More important, after sequence analysis, it was found that the HILDA gene is identical to the human LIF gene that has been just cloned by the lab of Donald Metcalf (Australia) at the same year [6], which suggested that HILDA is LIF [14].

Given that both DIA and HILDA/LIF are able to maintain the growth of DA-1a, Austin Smith was wondering whether HLDA/LIF can also inhibit the differentiation of mESC as DIA. The result was exciting. The medium conditioned by COS cells transected with HILDA/LIF cDNA clone could maintain typical mESC growth without biological or morphological differentiation.

 In view of the similarity between DIA and HILDA/LIF, it could be postulated that DIA is LIF [13].

At the same period, the lab of Donald Metcalf (Australia) which identified mouse and human LIF was also aware of the similarity between DIA and LIF. They have confirmed that LIF whether from human or mouse source was able to maintain the undifferentiated state of mESC [15]. This study and the two studies mentioned above that were done by Genetics Institute were published in the same issue on Nature in 1988. In other words, three articles related to LIF were published simultaneously in the same issue of one of the top international journals and all of them were discussing the effect of LIF on the culture of mESC, which indicated the importance of LIF in the maintenance of mESC in culture.

By the end of 1998 shortly after the human LIF has been cloned, the attention was turned to the roles of LIF in the culture of mESC.

Human LIF vs. Mouse LIF

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