Induction of Tumor Cell Differentiation

Differentiation (2000) 65:241–245

© Springer-Verlag 2000

MODELS & HYPOTHESES

Reed A. Flickinger

Induction of tumor cell differentiation

Accepted in revised form: 10 February 2000

Abstract It is proposed that cell proliferation with reduced individual cell growth (total protein accumulation) is necessary, but not sufficient, for cell differentiation. These conditions may facilitate transcription and accumulation of histones H1 and/or H1o relative to the core histones. This may have a critical role in cell differentiation.

Introduction Induction of tumor cell differentiation is often preceded by cessation of proliferation, and this remains an attractive possibility for tumor therapy. This paper will examine cell proliferation and growth during differentiation of various normal and tumor cells. Cell growth is defined as an increase in individual cell mass or total protein, while proliferation refers to cell doubling. The differentiation of numerous cell-types is characterized by an initial reduction of cell growth, followed by cessation of proliferation. However, in certain glandular tissues, such as liver, differentiated cells continue proliferation. Furthermore, some partially differentiated tumor cells continue proliferating while normal cells of the same degree of differentiation do not. These exceptions to the generalization that cell proliferation with reduced cell growth leads to cell differentiation may mean that this type of unbalanced growth is necessary, but not sufficient, for cell differentiation. The effect of reduced cell growth during a period of continued proliferation upon the accumulation of histone H1 and/or H1o will be examined. Evidence for a possible role of histone H1o in cell differentiation has been reviewed recently [65].

R.A. Flickinger Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14260, USA Present address: R.A. Flickinger P.O. Box 741, Captain Cook, Hawaii 96704, USA

Cell proliferation and growth prior to differentiation Normal cells decrease their cell growth while still proliferating prior to their differentiation. For example, synthesis of rRNA, which is indicative of their rate of protein synthesis [40], is reduced in myoblasts [27] and osteoblasts [52] during their final cell cycles before differentiation. Avian erythroid cell precursors cultured in vitro increase their rate of proliferation while reducing their cell size five-fold just before their differentiation [1]. Inducers of tumor cell differentiation usually inhibit cell growth initially, followed by a reduction in the rate of cell doubling. Mouse erythroleukemia (MEL) cells cultured with the inducer dimethylsulfoxide (DMSO) for 24 h reduce thir total RNA and protein by 15% and 13%, respectively [54]. However, there is no effect upon total DNA, indicating little change in cell proliferation. Retinoic acid (RA) induces differentiation of embryonal carcinoma cells. The volume of these cells decreases starting after 2 days with RA, but cell doubling time is increased after 3 days with RA [23]. Another study with this cell-type shows that RA inhibits protein synthesis during the first day, while DNA synthesis is unaffected [31]. Omission of an essential amino acid induces the differentiation of human promyelocytic leukemia (HL-60) cells [44]. Similar amino acid deprivation of normal human lymphocytes shows a much greater inhibition of RNA and protein synthesis than of DNA [2]. MEL cells induced to differentiate by hexamethylene bisacetamide (HMBA) or sodium butyrate have a marked reduction of cell volume which occurs prior to a delay in cell doubling time [13]. Actinomycin D (0.5—5 ng/ml) induces MEL differentiation, and RNA and protein synthesis are inhibited more than DNA synthesis [57]. After induction of differentiation for 5 days, the cells cultured in actinomycin D (1.5 ng/ml) have the same cell density as the controls. These results indicate that cell differentiation is characterized by an early reduction in the rate of cell growth, followed later by a decrease in the rate of cell proliferation, then by cell differentiation. This sequence of events is determined by the fact that cell growth drives cell pro-

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liferation, not vice versa [21, 42, 45]. The necessity of at least several cell cycles with reduced cell growth for cell differentiation is demonstrated by the failure of DMSO to induce differentiation of quiescent MEL cells [58]. In log phase cultures of MEL cells, the percent of cells differentiating is increased with additional cell proliferation [35]. However, additional cell proliferation with balanced cell growth-proliferation does not elicit cell differentiation. Cell doubling must occur with reduced cell growth. The granulocyte colony stimulating factor (GCSF) can promote differentiation of both normal and leukemic hematopoietic cells [34, 48]. Although G-CSF is mitogenic, it stimulates DNA synthesis ten times more than protein synthesis in cultured bone marrow macrophages [59]. This is cell proliferation with reduced cell growth, resulting in a kind of unbalanced cell proliferation-growth. Increasing the serum concentration stimulates proliferation, but not differentiation, of cultured MEL cells [7]. If cycloheximide is added to MEL cells cultured with a high serum level, over two thirds of the cells differentiate [7]. Although cyclohex-mide reduces the rate of proliferation at the higher serum level, the higher serum level drives cell doubling with a reduced level of protein synthesis. G-CSF is mitogenic for myelomonocytic leukemia cells and causes 15% of the cultured cells to differentiate [30]. RA alone induces 50% of the cells to differentiate, but used together G-CSF and RA induce 89% of the cells to differentiate [30]. Combined use of a mitogenic hematopoietic growth factor and RA to inhibit protein synthesis also induces differentiation of cultured acute myelogenous leukemia cells [38, 50, 60]. RA (100 nM) inhibits DNA synthesis of HL-60 leukemia cells, but the addition of mitogenic hematopoietic growth factors can obviate some of the RA inhibition [49]. RA elicits the appearance of hypophosphorylated retinoblastoma tumor suppressor protein (pRB) after 2 days of treatment in HL-60 cells [64]. This would inhibit RNA polymerases I [5] and III [61], which would down-regulate rRNA and tRNA synthesis, respectively. Cell density of the RA-treated cells is not reduced after 48 h, while the appearance of hypophosphorylated pRB in this same period indicates a reduced level of cell growth [64].

Unbalanced growth and histones H1 and H1o The period of reduced protein synthesis and continued cell proliferation is one of unbalanced growth. A number of reports indicate that this may allow the accumulation of histones H1 and/or H1o relative to the core histones. Reduced protein synthesis due to starvation of the mold Physarum causes the accumulation of histones H1 [18] and H1o [63] prior to the induction of differentiation. As indicated previously, inducers of tumor cell differentiation initially reduce rRNA and protein synthesis, followed later by cessation of proliferation. Downregulation of total protein synthesis facilitates accumulation of histone H1o [47]. Treatment with an inhibitor of

protein synthesis, cycloheximide, can induce transcription of H1o mRNA in MEL cells [47]. While reduction of total protein synthesis may be permissive for cell differentiation, various molecules that stimulate protein synthesis in an unregulated manner cause neoplastic transformation. Overexpression of the eukaryotic translation factor eIF4E induces transformation of 3T3 and CHO cells [56]. Other cell-types can be transformed by overexpression of insulin-like growth factor 2 (IGF-2) [37] or the IGF-1 receptor [24], both of which promote protein synthesis. Rosenwald [46] has proposed that deregulation of protein synthesis by oncogenes is the primary cause of tumor formation. Neoplastic transformation may be linked to a type of unbalanced growth in which there is an increased rate of unregulated protein synthesis relative to the rate of cell proliferation. This situation leads to the prediction that this would cause a decrease in histone H1 or H1o relative to the core histones. In this regard transformation by a ras oncogene results in a reduction of histone H1o relative to the core histones [28]. Reduction of protein synthesis resulting in the accumulation of histone H1o may not result in cell differentiation. Quiescent cultured neuroblastoma cells, which have reduced their level of protein synthesis, accumulate histone H1o [43]. However, these cells do not differentiate. One possible explanation for this failure to differentiate could be that incorporation of the H1o histone protein into chromatin requires a sufficient number of cell cycles. Transcription of the histone H1o gene is potentiated by DNA replication [14, 16]. Cells entering the quiescent state may not have undergone enough cell proliferation under conditions of reduced cell growth for cell differentiation. It seems that inhibition of protein synthesis alone does not induce cell differentiation. For example, rapamycin selectively inhibits the synthesis of growth-related proteins, but fails to induce cell differentiation [51]. This may be due to an early arrest of the cell cycle, thereby not allowing sufficient proliferation during the time of inhibition of protein synthesis.

Experimental induction of unbalanced growth The type of unbalanced growth proposed to be required for cell differentiation can be achieved by inhibiting cell growth more than cell proliferation or stimulating proliferation more than cell growth. This would increase the rate of cell proliferation relative to the level of protein synthesis. This is believed to increase the accumulation of histone H1 or H1o relative to the core histones [12]. One means of achieving a state of unbalanced growth permissive for cell differentiation would be to attempt simultaneous reduction of protein synthesis while maintaining cell proliferation. Protein synthesis is reduced in serum-starved quiescent cells. However, such cells can be induced to proliferate if injected with the cDNA for the E2FI transcription factor [20]. This factor is known to regulate various enzymes necessary for entry into the

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S phase. The idea that unbalanced growth is permissive for cell differentiation could be tested by overexpressing E2FI in cultured serum-starved tumor cells. However, some other means of reducing cell growth, while maintaining cell proliferation, would be required for achieving this type of unbalanced growth in vivo. In Drosophila imaginal disc cells ovrexpression of dmyc, a homolog of mammalian c-myc, produces larger cells, while reduced expression results in smaller cells [22]. There is no change of cell doubling time. These results suggest that discovery of an inhibitor of c-myc might allow cell proliferation with reduced protein synthesis, which would create a permissive condition for tumor cell differentiation. The regulation of the rate at which tumor cells proliferate in culture can be controlled by varying the serum concentration. Aside from the presence of the wellknown growth factors in serum, lysophosphatidic acid (LPA) and spingosine PO4 account for much of the mitogenic activity of serum [15, 39]. LPA can induce differentiation of cells of a human embryonic kidney cell line [29]. Mitogens can stimulate tumor cell proliferation, e.g., fibroblast growth factor (FGF) for pancreatic cancer cells [8] and hepatic growth factor for human melanoma cells [25]. Most mitogens, including serum, stimulate cell prolifration and growth equally. A mitogen that induces cell differentiation may stimulate proliferation more than cell growth. An inhibitor of cell growth (protein synthesis) may be required to obtain the desired kind of unbalanced growth in the cell cultures with high levels of serum. Retinoic acid (RA) has a number of advantages as an inhibitor of cell growth. It inhibits protein synthesis earlier than cell proliferation [23, 31], reduces the phosphorylation of pRB [64] and may prevent binding of the stimulating transcription factor UBF to regulatory sequences of the rRNA genes [11]. Both the latter two events would inhibit rRNA synthesis and, therefore, protein synthesis. Most importantly, ligand-bound RA receptors activate the promoter of the histone H1o gene [3, 4, 9, 33]. Some of the disadvantages of RA are that RA does not affect solid tumors [55] and tumor cells often have fewer RA receptors [41]. However, these disadvantages may be overcome. RA, used together with interferon α, can inhibit the proliferation of tumor cells not affected by RA alone [32]. Interferons can induce or increase the number of RA receptors and can restore RA-sensitivity to insensitive cell lines [10, 36, 62]. Both RA [17] and interferons [19] induce differentiation of a variety of tumor cells. However, used together they are more effective [32]. RA allows interferon to induce PKR, a doublestranded RNA-dependent protein kinase, which downregulates synthesis of certain proliferation-growth proteins [53]. While higher levels of RA inhibit cell proliferation, lower concentrations may stimulate cell division [6, 26]. HL-60 cells cultured with 0.1 µM RA for 2 days have 1.6 × more cells than in control cultures [6]. After 3 days of culture, proliferation is inhibited by RA. The results

reported suggest an experimental plan in which tumor cells would first be cultured in a medium with a high serum level and RA and interferon α for 1–2 days. The concentrations of RA and interferon α would be low enough to allow cell proliferation with reduced cell growth. After 1–2 days, the cells would be transferred to a new medium with less serum and more RA and interferon α, which would further decrease protein synthesis and inhibit cell proliferation. During the first step of this test additional cell cycling [14, 16], together with activation of the histone H1o gene promoter [3, 4, 9, 33], may increase transcription of histone H1o mRNA. The second step of the procedure should gradually reduce the rate of cell proliferation and cell growth (protein synthesis), with the lower rate of translation increasing the accumulation of histone H1o [47]. The hypothesis proposes that cell proliferation with reduced cell growth establishes a permissive condition for cell differentiation. Discovery of the optimal way to attain this type of unbalanced growth may lead to a procedure for differentiation therapy of tumor cells. Accumulation of histone H1 or H1o relative to the core histones may not only allow cell differentiation but it has been proposed that it also may select a differentiation pathway in a multipotent progenitor cell [12].

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