GROWTH ARREST OF PROLIFERATING CELLS BY TRANSGLUTAMINASE INHIBITORS

Abstract: Proliferating B cells have a cytosolic Transglutaminase (TGase), which exhibits absolute requirement for Ca<++> for its activity. The molecular mass of this TGase is 85 kDa. A method for arresting the lymphocytic cell cycle progression in the G1-phase is described by the use of TGase inhibitors, monodansyl cadaverine (MDC) and 1-(5-aminopentyl)-3-phenylthiourea (PPTU). Reversal of this inhibition allows the lymphocytes to enter the S-phase of normal cell cycle progression. A monocytic cell strain exhibits arrest of cell growth and failure to enter S-phase upon treatment with TGase inhibitor.

Description: GRO TH ARREST OF PROLIFERATING CELLS BY TRANSGLUTAMINASE INHIBITORS The government may own certain rights in the present invention pursuant to National Institute of Health Grant GM-35483 and National Cancer Institute Grant CA-38751. The present invention relates generally to a novel method comprising the arresting of eukaryotic cell growth in the Gi-phase of the cell cycle by treatment with a transgluta inase inhibitor. DNA synthesis and ensuing cellular replication are thus impeded. More particularly, the present invention demonstrates a process by which normal and neoplastic B and T lymphocytes are treated in vitro with pharmacologically achievable concentrations of trans-glutaminase inhibitors to block entry of the lymphocytes into the S-phase of the cell cycle which causes a cessation of lymphocytic proliferation. TGases (transglutaminases) are a group of Ca "1- * ' - dependent enzymes which catalyze an acyl transfer reaction between peptide-bound glutaminyl moieties and primary amines. Proteins and polyamines may be covalently cross-linked to proteins(1). These enzymes are widely distributed in body fluids and cells and have been reported to perform various physiological functions such as gamma-epsilon cross-linking of the fibrin clot in normal plasma, erythrocyte membrane deformation resulting from aging or genetic disease and seminal fluid clot formation in rodents(2). Tissue TGase, an 80-kDa intracellular enzyme, found in a variety of cells and tissues has been implicated in linking of membrane proteins and then to affect the physical properties of biomembranes (3-5) . Cultured G 6 glioma cells were found to contain a 67 kDa TGase in an inactive form which was released upon cell stimulation (26) . It has also been reported that TGase activity of human blood lymphocyte lysates increases up to 10-15 fold within 10-15 min after conconavalin A (Con A) activation of the cells (6) . Long term (72 hour) activation of lymphocytes with Con A appears to reduce TGase activity. This has led to the speculation that the TGase may be involved in the initial stimulation of the lymphocytes and may play a role in their blastogenesis. Transglutaminase has been reported to participate in receptor-mediated endocytosis; at least in cultured cells, such as fibroblasts and normal rat kidney cells (7-11) . Several studies have demonstrated that TGase may be involved in receptor-mediated endocytosis of alpha 2 macroglobulin (7) , immunoglobulin (27) , insulin, LDL (28), shigella toxin (29) and possibly EGF (30, 31). Polyamines have been shown to be physiological sub¬ strates for tissue TGase (4, 5). Although certain forms of TGase, e.g. , those found in hair follicles, skin (keratinocytes) and plasma (Factor XIII) , have been assigned some biological role, very little is known about the physiological role of tissue TGase in human B lymphocytes. TGase inhibitor inhibits the induction of murine B lymphocytes to form clones of antibody forming plasma cells (6) . The cell cycle begins with the completion of one cell division and ends with the completion of the next division. The time gap, usually early interphase, between the completion of cell division and the beginning of DNA replication is called the G x -phase of the cell cycle. The period of DNA replication is the S-phase. Continuous cellular growth with general increases in all of a cell's structures and functional capacities occurs during G x - and S-phases. Deprivation of growth factor components usually arrests most of the cells in the Gi- phase, thus producing a partial synchronization of the cell cycle in that particular culture. When the deprivation or stress factor is removed, the cells enter the S-phase and normal proliferation is resumed. Little information is available on the role of transglutaminase during lymphocyte activation and proliferation. Growth factor-dependent B lymphocytes (BCGF) , readily obtainable, provide a means to study the function of TGase during cell cycle progression. Control of the transglutaminase function in lymphocytes and possibly other transformed cells, e.g. malignant monocytes, in late Gi-phase in the cell cycle can arrest cellular proliferation. This can provide a highly desirable adjunct to chemotherapy when used in combination with Gi-specific anti-neoplastic agents. Abbreviations used herein include: kDa kiloDalton BCGF B Cell Growth Factor MDC Monodansyl Cadaverine PPTU l-(5aminopentyl)-3-phenylthiourea 3 H-Tdr tritiated thymidine Ci Curie CPM counts per minute BSA bovine serum ablumin SDS-PAGE sodium dodecyl sulfate- polyacrylamide gel electrophoresis FACS fluorescent activated cell sorter FCS inactivated fetal calf serum TBS tris-buffered saline [lOmM Tri-HCl, 0.15M Sodium Chloride, bH7.5) SEM standard error of the mean. The present invention comprises a method for arresting cellular growth and thereby blocking DNA synthesis in cells by treating these same cells with a transglutaminase inhibitor. Monodansyl cadaverine and l- (5-aminopentyl)-3-phenylthiourea are two such transglutaminase inhibitors which successfully arrest cellular growth and block entry of the cells into the S- phase of the cell cycle. Other transglutaminase inhibitors may also effectively arrest cellular growth in the same manner. A number of different cell types, both neoplastic and non-neoplastic, have shown arrest of cellular growth upon treatment with a transglutaminase inhibitor in a concentration which was sufficient to inhibit the transglutaminase activity of the cell. BCGF dependent, B lymphocytes treated in vitro with aqueous solutions of increasing concentrations of monodansyl cadaverine (about 25 to about 200 micromolar) or l-(5-aminopentyl)-3- phenylthiourea (about 0.5 to about 2.0 millimolar) show an almost linear dose dependent inhibitory effect on the arrest of B lymphocytic cellular growth. Neoplastic B cell lines, such as B lymphoma cells derived from eight patients with B lymphoma, show arrested cell growth in the Gi-phase of the cell cycle upon treatment, in vitro, with a transglutaminase inhibitor, monodansyl cadaverine. Another neoplastic cell line which shows inhibition of cell growth jLn vitro when treated with a transglutaminase inhibitor, monodansyl cadaverine, was a T lymphocytic cell line derived from a patient with acute lymphocytic leukemia. Other neoplastic cell lines which exhibit in vitro arrest of cellular growth when exposed to a transglutaminase inhibitor, monodansyl cadaverine, are two myeloma cell lines and a monocytic cell line. All of the neoplastic cell lines mentioned were treated with concentrations of about 25 to about 200 micromolar monodansyl cadaverine. Therefore the present invention provides a novel means of arresting eukaryotic cell growth comprising treating the eukaryotes with a transglutaminase inhibitor in a concentration which is sufficient to inhibit the transglutaminase activity of the eukaryotic cells. Lymphocytes, both B and T, and monocytes have been tested and showed arrest of cellular growth when treated with one of several transglutaminase inhibitors, monodansyl cadaverine (about 25 to about 200 micromolar concentration) or l-(5-aminopentyl)-3-phenylthiourea (about 0.5 to about 2.0 millimolar concentration). The invention should not be necessarily limited to these specific cell types or cell lines nor to these specific transglutaminase inhibitors. Figure 1A demonstrates the normal proliferation of BCD4 cells by monitoring the incorporation of 3 H-Tdr in varying amounts of cells in the presence of serial dilutions of BCGF. BCD4 cells at 5xl0 3 cells/well (solid circles) , lOxlO 3 cells/well (solid squares) , 15xl0 3 cells/well (open circles) , 20xl0 3 cells/well (open squares) are plotted against BCGF concentrations. Figure IB indicates normal growth characteristics for BCD4 cells as average CPM for 3 H-Tdr incorporation plotted as a function of cells per well at 10% (v/v) BCGF (open triangles) 5% (v/v) BCGF (solid circles) , and at 1.2% (v/v) BCGF (solid triangles). Figure 2A, a histogram, shows that the BCD4 cell contained a significant level .of transglutaminase activity and that this activity was completely Ca^ dependent. This absolute requirement for Ca** affirmed the enzyme activity of the B cell lysate as transglutaminase. Figures 2B and 2C further confirm the presence of transglutaminase in the proliferating B cell lysate. Cytosolic proteins were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. Coomasie blue staining of the nitrocellulose strip indicated the presence of several different proteins (Figure 2B., Lane 1) . A duplicate nitrocellulose strip, used for immuno- blot analysis, showed the presence of a single 85-kDa protein (Figure 2C, Lane 1), which was recognizable by a monoclonal antibody against guinea pig liver tissue transglutaminase (Figure 2C. , Lane 2) . Figure 3A represents a P-100 gel permeation chromatogram of the cytosolic extract of BCD4 cells. Protein concentration, measured at A 280 (open circles) and TGase activity, CPM incorporated (solid circles) , are plotted against fraction number. Figure 3B is a Western Blot analysis of the P-100 column fractions of BCD4 cell lysate and indicates the presence of a single immunoreactive band at 85kDa in those fractions where transglutaminase activity was observed. Figure 4A demonstrates the cytosolic origin and Ca " " " dependence of the TGase found in BCD4 cells (open circles) . No detectable amount of TGase activity was found in the membrane fraction from the BCD4 cells (solid circles) . Figure 4B and 4C. Figure 4B indicates the presence of multiple protein bands in cytosolic (lane 1) and membrane (lane 2) preparation from BCD4 cells yet only a single immunoreactive band is present in the cytosolic fraction of the BCD4 cells (Figure 4C, lane 1) . Figure 5 shows the blocked BCGF-induced proliferation of BCD4 cells and the dose dependent monodansyl cadaverine mediated inhibition of the BCD4 cells. Average CPM of triplicate data points are plotted as a function of BCGF concentration in the absence (solid circles) or presence of 25.0 uM (solid squares), 50.0 uM (open circles), 100.00 uM (open triangles), and 200.00 uM (open squares) MDC. The inset shows CPM incorporated as a function of MDC concentration at 10.0% (v/v) BCGF (solid circles), 5% (v/v) BCGF (open circles), 2.5% (v/v) BCGF (solid squares), and 1.2% (v/v BCGF (open triangles) . Figure 6 shows the blocked, BCGF-induced, prolifera¬ tion of BCD4 cells and the dose dependent l-(5 aminopen- tyl)-3-phenylthiourea(PPTU) mediated inhibition of the BCD4 cells. Interestingly, the rate of PPTU induced inhibition was higher at a lower concentration of BCGF compared to a higher concentration of BCGF. This was not observed with MDC. BCD4 cells were cultured in absence (solid circles), or presence of 0.5 uM (open circles), 1.0 uM (open triangles) or 2.0 uM (open squares) PPTU. Average of triplicate CPM are plotted as a function of BCGF concentration (v/v) at each PPTU concentration. The inset shows the dose-dependent inhibitory effect of PPTU at 5% (v/v) BCGF (open triangles) and 1.2% BCGF (solid circles) . Figure 7 indicates that complete inhibition of cell proliferation by MDC, as measured by 3 H-Thymidine incorporation, occurred up to 16 hours after initiation of culture. Addition of MDC after 24 hours allowed about 30% of the cells to progress to S-phase. If MDC is not added to the BCD4 cell culture until 32 hours after initiation then about 60% of the cells progress to the S- phase. BCD4 cells were cultured with either 5% (v/v) BCGF (open circles), or 2.5% (v/v) BCGF (solid circles) and 1.0% Nutridoma. Figure 8 shows the FACS analysis of proliferating B lymphocytes cultured in the presence or absence of 200 micromolar MDC. A) Random B cell population DNA content cultured in the absence of MDC and B) B cell population DNA content cultured in the presence of 200 micromolar MDC. This clearly indicates that B cells cultured in presence of 200 micromolar MDC accumulated in G x phase. A method for arresting the growth of lymphocytes in the Gi-phase of the cell cycle, thereby preventing entry into the S-phase by treatment with transglutaminase inhibitors, is demonstrated herein. The transglutaminase activity in the proliferating lymphocytes was found in the cytosolic fraction and exhibited absolute requirement for Ca " " " for its catalytic activity. Monoclonal antibodies against liver tissue transglutaminase detected an 85 kDa protein band from the BCGF dependent lymphocyte lysate which correlated with the cytosolic transglutaminase activity. Experiments conducted with two different transglutaminase inhibitors, monodansyl cadaverine and 1- (5-aminopentyl)-3-phenyl-thiourea, clearly indicated that B lymphocyte proliferation, as monitored by 3 H-thymidine incorporation, was blocked by both inhibitors. This blockage was dose-dependent. Complete blockage was obtained with pharmacologically achievable concentrations of both inhibitors. Further experiments, utilizing eight B lymphoma cell lines, one T acute lymphocytic leukemia cell line, two myeloma cell lines and one monocytic cell line, also arrested cell proliferation in the G x -phase when exposed to monodansyl cadaverine. Reversal of monodansyl cadaverine-induced blockage of the cell cycle demonstrated that this inhibitor was not toxic to the cells. B cells arrested in Gi-phase by MDC treatment for as long as 48 hours can continue their progression to S-phase after removal of the transglutaminase inhibitor. These examples are presented to describe preferred embodiments and utilities of the present invention and are not meant to limit the present invention unless otherwise stated in the claims appended hereto. EXAMPLE 1 The human B cell line, BCD4, used in this study was derived from anti-IgM activated B cells isolated from normal human peripheral blood lymphocytes. These cells have been proliferating in an exogenously supplied, BCGF dependent, manner for more than a year (16) . The doubling time for BCD4 cells is 36-40 hours. B lymphocyte proliferative assay: The microculture assays were performed in 96-well flat bottom icrotiter plates (16) . BCD4 cells were washed and each microwell of the microtiter plate was filled to a final volume of 200 uL of RPMI 1640 medium, supplemented with 1% nutridoma, each containing either 5, 10, 15 or 20 x 10 3 cells/well in the presence of V/V serial dilutions of BCGF. The microcultures were pulsed for the last 16 hours of the 40 hour culture period with 0.1 uCi/well of 3 H-Tdr (6Ci/m mole). The cultures were harvested by collecting the cells on the glass fiber filter paper and the radioactivity was determined by scintillation counting. In figure 1A the average CPM for 3 H-Tdr incorporation in BCD4 cell from triplicate data points was plotted against BCGF concentration. Figure IB. shows the average CPM from triplicate data points of 3 H-Tdr incorporation as a function of cells/well at varying concentrations of BCGF. This resultant data assured the use of microculture assays for monitoring entry of BCD4 cells into S-phase. Presence of TGase in proliferating B cells: In order to determine the functional role of TGase in the cell cycle progression, the presence of the enzyme in the BDC4 cells must be demonstrated. For this purpose, the cell lysate, of factor dependent BDC4 cells, was assayed for TGase activity. Transglutaminase activity assay: TGase activity in cell extracts was determined in triplicate as a Ca " " " dependent incorporation of 3 H-Putres- cine into dimethylcasein (14, 15). The cells were washed with TBS (Tris buffered saline, pH 7.5) and the final cell pellet, containing 6-9 million cells, was lysed by a brief sonication in 0.3 uL of TBS containing 1.5uM beta- mercaptoethanol. The assay mixture (final volume 100 uL) contained 2% dimethylcasein, 5uM CaCl 2 , 20uM dithiothreitol and 50 uL of the cell lysate in 15uM Tris- buffered saline (pH 7.5). The assay was carried out at 37"C for the appropriate time period and at predetermined time intervals the aliquots (25 uL) were removed, placed -li¬ on 1 cm 2 of Whatmann 3MM filter paper, and immersed immediately in ice cold 10% trichloroacetic acid (TCA) for 5 minutes. The filters were then washed two more times with 5% TCA for 10 minutes each washing and once with ethanol and dried. The radioactivity incorporated was determined by scintillation counting in a Beckman Tri-Carb scintillation counter. The histogram in Figure 2A shows that BCD4 cells contained a significant level of TGase activity, and that this activity was completely Ca^ dependent. Another divalent ion, such as Mg "1-1" , could not substitute for Ca "1-1" in the assay. This absolute requirement for Ca "1"1' established that the enzyme activity in the cell lysate was indeed due to TGase. In order to further confirm the presence of TGase in the cell lysate of proliferating B cells, cytosolic proteins were fractionated by SDS-PAGE and then transferred onto a nitrocellulose membrane for immunoblotting as described in the following section. SDS-polvacrylamide gel electrophoresis and immunoblot analysis: Cell lysates, fractionated in two identical sets on a 10-12% polyacrylamide slab gel (18 cm x 13 cm x 0.15 cm) under denaturing and reducing conditions using an SDS-Tris-Glycine buffer system (19) were transferred onto a nitrocellulose paper (20) . One set of samples was stained on the nitrocellulose paper by amido black B. The other identical set of protein samples was used for immunoblotting (21) . Briefly, the nitrocellulose filter was saturated with bovine serum albumin (BSA) for a minimum of 2 hours. Subsequently the filter was washed extensively with TBS and incubated with mouse-monoclonal anti-TGase (12) for 1 hour. The excess antibody was washed out and the filter was exposed to alkaline phosphatase, conjugated to rabbit anti-mouse IgG, for 30 minutes. Excess rabbit antibody was washed away and the filter was exposed to substrate according to the manufacturer's instructions (Promega, Madison, WI) . The reaction was stopped by washing off the substrate after the bands were fully developed, which usually occurred within 15 minutes. Coomasie blue staining of the nitrocellulose strip indicated the presence of several different proteins (Fig. 2B, Lane 1) . A duplicate nitrocellulose strip, used for immuno-blot analysis, showed the presence of a single 85-kDa protein (Fig. 2C. , Lane 1) , recognizable by a monoclonal antibody against guinea pig liver tissue TGase (Fig. 2C, Lane 2). Fractionation of TGase on P100: The enzyme activity was further characterized by fractionating the BCD4 cell lysate on a P-100 gel permeation column. One and a half milliliters of cytosolic BCD4 cell extract prepared from 10 8 BCD4 cells was loaded on a P-100 gel filtration column (75 cm X 1.6 cm) pre-equilibrated in TBS. One milliliter fractions were collected from the column and protein elution was monitored by absorbance at 280 nm. Fractions numbered 17 through 56 were tested for TGase activity. Protein concentration (A 280 ) and TGase activity are plotted against fraction number in Figure 3A. The major peak of TGase activity was present in Fraction 22 which represented some slightly included material. Denoted column fractions were separated on a 10% polyacrylamide gel under reducing and denaturing conditions. Proteins were transferred onto a nitrocellulose paper and immunoblotted with Mab against TGase. The Western blot analysis of these same column fractions indicated the presence of a single immunoreactive band at 85 kDa in only those fractions where TGase activity was observed (Figure 3B) . The enzyme activity in individual fractions, roughly, corresponded to the amount of enzyme protein as shown by im unoblot analysis. Intracellular localization of lymphocytic TGase: Several reports have shown that a part of TGase activity is associated with the plasma membrane domains (22,23). Membranes were prepared by sucrose density flotation method (17) . BCD4 cells were lysed in lysing buffer (18) , and the lysate was centrifuged on sucrose step density gradients for 75 minutes at 90,000 x g to separate membranes from nuclei and soluble cytosolic proteins. The membrane proteins were solubilized by 0.2% Nonidet P-40. The purified membrane fraction and the soluble cytosolic fraction were tested for the presence of TGase activity at various Ca 'H" concentrations. Material equivalent to 3 x 10 6 BCD4 cells was used for each assay point. As shown in Figure 4A. , there was no detectable amount of TGase activity in the membrane frac¬ tion. However, the cytosolic fraction showed a significant level of enzyme activity that was Ca " " " concentration dependent. Proteins from the cytosolic and the membrane preparations were fractionated in two identical sets on 10% polyacrylamide gel under reducing and denaturing conditions and transferred to nitrocellulose membranes. The Coomassie blue staining of the nitrocellulose strip containing the fractionated cytosolic proteins (Fig. 4B, Lane 1) and membrane proteins (Fig. 4B, Lane 2) revealed the presence of several different proteins. A duplicate nitrocellulose strip was immunoblotted with mouse anti-TGase antibody and developed with substrate for alkaline phosphatase which was conjugated to a second antibody. The cytosolic fraction which showed the presence of enzyme activity, also exhibited presence of a 85-kDa immunoactive band (Fig. 4C, Lane 1) , whereas the membrane fraction, which lacked enzyme activity, also showed no detectable band at 85-kDa (Fig. 4C, Lane 2) by im unoblot technique. Inhibitory effect of monodansyl cadaverine (MDC) : In order to determine if TGase has any functional role in the B lymphocyte proliferation, the effect of monodansyl cadaverine (MDC) on the entry of the cells into S-phase was then tested. Thoroughly washed BCD4 cells were plated (in triplicate) 20 X 10 3 cells per well in a final volume of 200 uL of RPMI 1640 medium supplemented with 1% Nutridoma, serial dilutions of BCGF and various concentrations of monodansyl cadaverine (0, 25, 50, 100 or 200 uM) . The microcultures were labelled with 0.1 uCi of 3 H-Tdr (6Ci/m mole) for the last 16 hours of the 40 hour culture period. The 3 H-Tdr incorporation was determined by counting the harvested cells in a scintillation counter. Average CPM of triplicate data points were plotted as a function of BCGF concentration in the absence or presence of monodansyl cadaverine (Figure 5.) The inset of Figure 5. shows the CPM incorporated as a function of monodansyl cadaverine (MDC) concentration at 1.2, 2.5, 5.0 and 10.0% BCGF (V/V). Monodansyl cadaverine (MDC) , a potent pseudo substrate of TGase (24) , blocked BCGF-induced proliferation of the BCD4 cells (Figure 5.). MDC- mediated inhibition of B-cell growth was dose dependent (Figure 5., inset) and a complete inhibition was observed at 200 uM concentration of MDC. These results suggested that MDC was inhibiting DNA-synthesis either by blocking S-phase entry of or the progression of cells through S- phase. Proliferating B lymphocytes were cultured for 24-36 hours in the presence or absence of 200 uM MDC. The cells were washed, lysed and the nuclei stained with propidium bromide. Cell cycle analysis was performed by FACS. This analysis of MDC treated and untreated BCGF dependent B cells confirmed that the cells were blocked in G x phase (Figure 8) . Kinetics of MDC inhibitory effect on cell proliferation: In order to determine if TGase has any functional role in the B lymphocyte proliferation, the effect of monodansyl cadaverine (MDC) on the entry of the cells into S-phase was next tested. Factor starved BCD4 cells, in general are arrested in early Gi-phase and are partially synchronized. When these cells are exposed to BCGF, after extensive washing, their progression towards S-phase starts and the entry into S-phase can be monitored by 3 H-Tdr incorporation. If TGase is involved in the initial BCGF signal transduction, delayed exposure of these cells to MDC should have no effect on their entry into S-phase. Therefore, experiments were conducted where sets of B cell microcultures were initiated at zero time with the addition of BCGF. 2 x 10 3 BCD4 cells per well were cultured for 48 h in 200 uL of RPMI 1640 medium supplemented with either 5% (v/v) BCGF (open circles) , or 2.5% (v/v) BCGF (solid circles) and 1.0% Nutridoma. The cultures were labelled with 0.1 uCi per well of 3 H-Tdr (6.0 Ci per mmole) for last 24 hours and at the indicated time points MDC was added to final concentration of 200 of uM. Harvested cultures were counted in a scintillation counter to determine levels of 3 H-Tdr incorporation. Each point represents average of triplicate numbers. Results of these experiments (Fig. 7) indicated that complete inhibition of cell proli¬ feration, as measured by 3 H-Thymidine incorporation, oc¬ curred even when MDC was added 16 hours after the initiation of culture. When MDC was added 24 hours after the initiation of culture 30% of the cells progressed to s-phase and therefore incorporated 3 H-Thymidine. In 32 hours, about 60% of the cells were in S-phase as determined by 3 H-Thymidine incorporation. Reversal of MDC inhibition: Polyamines are the known physiological substrates for transglutaminase in lectin-activated lymphocytes (6) . If MDC was competing with these natural substrates, the inhibitory effect of MDC would be reversible. Also, it was important to show that the MDC-mediated inhibition of B cell growth should be due to growth arrest rather than due to the toxic effect of MDC on B cells. The cell cycle progression of the cells which had been blocked with 100 uM or 200 uM MDC for 36 hours was monitored. After removal of MDC, the blocked cells appeared to continue their progression through S-phase in the absence of BCGF (Table I) . In contrast, the cells which were continuously exposed to MDC, could not enter the S-phase. The washed cells, which were never exposed to MDC, also did not proceed to S-phase in the absence of BCGF. TABLE I Reversal of Inhibitory Effect of MDC Presence of MDC 3 H-Thvmidine Incorporated (cpm)# During During Experiment Experiment Preincubation Microculture I II + + B cells were incubated in complete medium (RPMI 1640, 5% FCS, 10% BCGF) with 100 uM of MDC (Exp I) and 200 uM MDC (Exp II) for 48 h. At the end of this incubation, cells were washed 2x with RPMI 1640 medium and used for testing S- phase entry in microculture assay. Microcultures were performed in triplicate in 200 uL of RPMI 1640 supplemented with 5% FCS without BCGF. Each well contained 20,000 B cells (Exp I), 10,000 B cells (Exp. II). + indicate presence of MDC; - indicates absence of MDC. # Numbers represent average of triplicate values. SEM in each case was less than 10%. Inhibitory effect of monodansyl cadaverine on various malignant cell strains: Eight B lymphoma, two myeloma, one T and one monocytic leukemia cell strains were treated with monodansyl cadaverine in exactly the same manner as the BCD4 cells. Each cell strain culture was plated (in triplicate) 25 x 10 3 cells per well in a final volume of 200 uL of RPMI 1649 medium. Increasing concentrations of monodansyl cadaverine (0,100 and 200 uM) were added. Microcultures were labelled for 16 hours with 0.1 uCi/well of 3 H-Tdr (6Ci/mmol) . The 3 H-Tdr incorporation was determined by counting the harvested cells in a scintillation counter. Average CPM of triplicate data points were reported. Table II shows the inhibitory effects of monodansyl cadaverine on various types of malignant cell strains. Almost complete inhibition is observed with the 200 uM concentration level of MDC for all cell strains tested. It is believed that such TGase inhibition may be useful for the in vivo treatment of analogous malignant tumors. This treatment, when appropriate dosage schedules are elucidated, should inhibit such malignant cell growth and possibly render inhibited cells more susceptible to conventional chemotherapy and/or radiotherapy. TABLE II Patient MDC Concentration (Cell Type) (uM) Malignancy 100 200 TR 12600 a 4505 (64%) b 490 (96%) (B lymphoma) CLL (Chronic lymphocytic leukemia) RY 17510 4504 (74%) 1554 (91%) (B lymphoma) IBS (Immunoblastic sarcoma) TI 61120 39316 (36%) 3033 (95%) (B lymphoma) LCL (Large cell lymphoma) AJ 10490 701 (93%) 117 (99%) (B lymphoma) Burkitt's lymphoma MT 42800 1525 (96%) 74 (100%) (B lymphoma) Burkitt's lymphoma MI 33202 27298 (18%) 5217 (84%) (B lymphoma) LCL-transformed (Large cell lymphoma transformed) CJ 36541 7988 (78%) 844 (98%) (B lymphoma) TG 63312 56360 (11%) 2149 (96%) (B lymphoma) LCL (Large cell lymphoma) BC 61868 5082 (92%) 224 (99%) T-ALL (Acute lymphocytic leukemia) THP-1 6048 1185 (80%) 152 (97%) (monocytic leukemia) IgG producing myeloma 2916 706 (76%) 155 (95%) IgM producing myeloma 4162 1892 (55%) 492 (88%) Average of triplicate CPM values for 3 H-TDR incorporati in microculture assays. Each culture was set in 200 uL RPMI 1640 medium with 25,000 cells/well. Cultures were labelled for 16 h with 0.1 uCi/well of 3 H-Tdr (6Ci/mmol SEM was less than 10% in each case. Numbers in parenthesis indicate percent inhibition. EXAMPLE 2 Another known transglutaminase inhibitor, l-(5- aminopentyl)-3-phenylthiourea (PPTU), was tested. PPTU obtained from Dr. S. I. Chung (National Cancer Institute, Bethesda, MD) and synthesized according to Lee et al. (25) , has been shown to specifically inhibit the transglutaminase activity, in vivo, in injected animals (25) . The same human B cell line used in Example 1, BCD4, was tested with PPTU. Kinetics of PPTU inhibitory effect on cell proliferation: Sets of B cells cultured as described in Example I were initiated at zero time with BCGF in serial dilutions of 1.2 and 5% (v/v) . PPTU was added to the miσrocultures in final concentrations of 0.0, 0.5, 1.0 and 2.0 uM at various time points, in order to progressively reduce the total time of exposure of the BCD4 cells to the inhibitor. All cultures were pulse-labelled with 3 H-Tdr for the same length of time and were terminated at the same time. Averages of the triplicate CPMs were plotted as a function of BCGF concentration (v/v) at each PPTU concentration in Figure 6. The inset shows the dose dependent inhibitory effect of PPTU at 1.2 and 5% (v/v) of BCGF. A much higher concentration of PPTU than MDC was re¬ quired to achieve the same transglutaminase inhibitory effect. PPTU did prevent entry of the BCD4 cells into S- phase. Interestingly, the rate of PPTU-induced inhibition was higher at a lower BCGF concentration (1.2%) as compared to the inhibition ratio at higher BCGF concentration (5.0%). In the case of MDC, no such difference was observed. This observation was in agreement with the earlier observations that much higher concentrations of PPTU are needed to inhibit the TGase catalyzed clot formation than that of MDC (25) . 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Nigam, V.N., Morais, R. and Karasaki, S. 1971. A simpl method for the isolation of rat liver cell plasma membranes i idostanic sucrose. Biochim. Biophys.Acta 249:34-40. 18. Sahasrabuddhe, C.G., Morgan, J. , Sharma, S., Mehta, S., Martin, B., Wright, D. , Maizel A. 1984. Evidence for an intracellular precusor for human B-cell growth factor. Proc.Natl.Acad,Sci. (USA) 81:7902-7906. 19. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature (Lond) 227:680-685. 20. Brunette, W.N. (1981) "Western Blotting": Electro- phoretic transfer of proteins from sodium dodecyl sulfate- polyacrylamide gels to unmodified nitrocellulose and radio- graphic detection with antibody and radioiodinated Protein. Anal.Biochem. 112:195. 21. Sahasrabuddhe, C.G., Martin, B.A., David, F.M. (1988) Immunological evidence for the relation between low M r secret form of human B cell growth factor and an intracellular 60k protein. Lymphokine Res. 7(2) :141-155. 22. Slife, C.W. , Dorsett, M.D., and Tillotson, M.L. (1986) Subcellular location and identification of large molecular weight-substrate for the liver plasma membrane transgluta¬ minase. J.Biol.Chem. 261:3541-3456. 23. Tyrrell, D.J. , Sale, W.S., and Slife, C.W. (1986) Localization of liver transglutaminase and a large molecular weight transglutaminase substrate to a distinct plasma domai J.Biol.Chem. 261:14833-14836. 24. Lorand, L. , Rule, N.G., Ong, H.H., Furlanetto, R. , Jacobson, A., Downey, J. , Oner, N. , and Bruner-Lorand, J. (1968) Biochemistry 7:1214-1223. 25. Lee, K.N., Fesus, L. , Yancey, S.T., Gerard, J.E., and Chung, S.I. (1985) Development of selective inhibitors of transglutaminase. J.Biol.Chem. 260:14689-14694. 26. Korner, G., and Bachrach, U. (1985) Activation and de novo synthesis of transglutaminase in cultured glioma cells. J.Cell Physiol. 124:379-385. 27. Leu, R.W. , Herriot, M.J. , Moore, M.P., Orr, G.R., and Birchbichler, P.J. (1982) Enhanced transglutaminase activity associated with macrophage activation. Possible role in Fc- mediated endocytosis. Exp. Cell Res. 141:191-199. 28. Pastan, I.H., Willingham, M.D. (1981) Journey to the center of the cell: role of receptosomes. Science 214:504- 29. Keusch, G.T. (1981) Receptor-mediated endocytosis of shingella cytotoxin. In Receptor Mediated Binding and Internalization of Toxins and Hormones. J.L. Middlebrook and L.D. Kohn, eds. Academic Press. New York p.95. 30. Haigler, H.T., Willingham, M.C., and Pastan, I. (1980) Inhibitors of 125 I-epidermal growth factor internalization. Biochem.Biophys.Res.Commun. 94:630-637. 31. Haigler, H.T. , Maxfield, F.R., Willingham, M.C., and Pastan, I. (1980) Dansylcadaverine inhibitors internalizatio of 125 I-epidermal growth factor in Balb 3T3 cells. J.Biol.Chem. 255:1239-1241. Changes may be made in the TGase inhibitors described herein or in the steps or the sequence of steps of the method described herein without departing from the concept and scope of the invention as defined in the following claims.

Claims: CLAIMS : 1. A method for arresting the growth of eukaryotic cells comprising inhibiting transglutaminase activity of said cells by treatment with a transglutaminase inhibitor. 2. A method for arresting lymphocyte growth comprising treating lymphocytes with a transglutaminase inhibitor in an amount sufficient to inhibit transglutaminase activity of the lymphocytes. 3. A method for arresting B lymphocyte growth comprising treating B lymphocytes with a transglutaminase inhibitor in an amount sufficient to inhibit transglutaminase activity of said B lymphocytes. 4. A method for arresting T lymphocyte growth comprising treating said T lymphocytes with a transglutaminase inhibitor in an amount sufficient to inhibit transglutaminase activity of said T lymphocytes. 5. A method for arresting monocytes in a cell cycle Gi-phase comprising treating said monocytes with a trans¬ glutaminase inhibitor in an amount sufficient to inhibit transglutaminase activity of said monocytes. 6. The method of claim 1, 2, 3, 4, or 5 wherein the transglutaminase inhibitor is monodansyl cadaverine. 7. The method of claim 1, 2 or 3 wherein the transglutaminase inhibitor is l-(5-aminopentyl)-3- phenylthiourea. 8. A process for arresting in vitro growth of lymphocytes comprising treatment with a transglutaminase inhibitor in an amount sufficient to inhibit transglutaminase activity of said lymphocytes. 9. The process of claim 8, wherein the transglutaminase inhibitor is monodansyl cadaverine. 10. The process of claim 8, wherein the transglutaminase inhibitor is l-(5-aminopentyl)-3-phenylthiourea. 11. The method of claim 1, wherein the eukaryotic cells are inhibited by treatment with a solution comprising about 25 micromolar to about 200 millimolar transglutaminase inhibitor. 12. The method of claim 2, 3 or 4, wherein the lymphocytes are treated with a solution comprising about 25 micromolar to about 200 millimolar transglutaminase inhibitor. 13. The method of claim 5, wherein the monocytes are treated with a solution comprising about 25 micromolar to about 200 millimolar transglutaminase inhibitor. 14. The method of claim 1, wherein the eukaryotic cells are inhibited by treatment with a solution comprising about 25 to about 200 micromolar monodansyl cadaverine. 15. The method of claim 2 , 3 or 4, wherein the lymphocytes are treated with a solution comprising about 25 to about 200 micromolar monodansyl cadaverine. 16. The method of claim 5, wherein the monocytes are treated with a solution comprising about 25 to about 200 micromolar monodansyl cadaverine. 17. The method of claim 1, wherein the eukaryotic cells are inhibited by treatment with a solution comprising about 0.5 to about 2.0 millimolar l-(5-aminopentyl)-3- phenylthiourea. 18. The method of claim 2 or 3, wherein the lymphocytes are treated with a solution comprising about 0.5 to about 2.0 millimolar l-(5-aminopentyl)-3-phenylthiourea. 19. The process of claim 8, wherein treatment comprises exposure of the lymphocytes to a solution comprising about 25 micromolar to about 200 millimolar transglutaminase inhibitor. 20. The process of claim 8, wherein treatment comprises exposure of the lymphocytes to a solution including about 25 to 200 micromolar monodansyl cadaverine. 21. The process of claim 8, wherein treatment comprises exposure of the lymphocytes to an aqueous solution including about 0.5 to 2.0 millimolar l-(5-aminopentyl)- 3-phenylthiourea. 22. A process for arresting the growth of B lymphocytes, in vitro, comprising the treatment of said B lymphocytes in culture with a transglutaminase inhibitor in an amount sufficient to inhibit transglutaminase. 23. The process of claim 22, wherein the B lymphocytes are treated with a solution comprising about 25 micromolar to about 200 millimolar transglutaminase inhibitor. 24. The process of claim 22, wherein the B lymphocytes are treated with an aqueous solution comprising about 25 to about 200 micromolar monodansyl cadaverine. 25. The process of claim 22, wherein the B lymphocytes are treated with a solution comprising about 0.5 to about 2.0 millimolar l-(5-aminopentyl)-3-phenylthiourea. 26. A process for arresting in vitro growth of T lymphocytes in the Gi cell cycle phase comprising treatment of said T lymphocytes in culture with a transglutaminase inhibitor in an amount sufficient to inhibit transglutaminase activity. 27. The process of claim 26, wherein the transglutaminase inhibitor is monodansyl cadaverine. 28. The process of claim 26, wherein the T lymphocytes are treated with an aqueous solution comprising about 25 to about 200 micromolar monodansyl cadaverine. 29. The process of arresting in vitro growth of monocytes comprising treatment of said monocytes in culture with a transglutaminase inhibitor in a sufficient amount to inhibit transglutaminase activity. 30. The process of claim 29, wherein the transglutaminase inhibitor is monodansyl cadaverine. 31. The process of claim 29, wherein the monocytes are treated with an aqueous solution comprising about 25 to about 200 micromolar monodansyl cadaverine. 32. A method for blocking DNA synthesis in lymphocytic cells, comprising treating said cells with a concentration of transglutaminase inhibitor sufficient to block said synthesis. 33. A method for blocking DNA synthesis in monocytes comprising treating said monocytes with a concentration of transglutaminase inhibitor sufficient to block said synthesis.

Inventors: MEHTA KAPIL, SAHASRABUDDHE C G, MEHTA, KAPIL, SAHASRABUDDHE, C., G.
Applicants: UNIV TEXAS, BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM
Application year: 1990
Main classification: A61K31
IPC codes: A61K31, A61K45, A61P35, A61P43