Tissue transglutaminase and apoptosis: sense and antisense transfection studies with human neuroblastoma cells.

In this report, we show that the overexpression of tissue transglutaminase (tTG) in the human neuroblastoma cell line SK-N-BE(2) renders these neural crest-derived cells highly susceptible to death by apoptosis. Cells transfected with a full-length tTG cDNA, under the control of a constitutive promoter, show a drastic reduction in proliferative capacity paralleled by a large increase in cell death rate. The dying tTG-transfected cells exhibit both cytoplasmic and nuclear changes characteristic of cells undergoing apoptosis. The tTG-transfected cells express high Bcl-2 protein levels as well as phenotypic neural cell adhesion molecule markers (NCAM and neurofilaments) of cells differentiating along the neuronal pathway. In keeping with these findings, transfection of neuroblastoma cells with an expression vector containing segments of the human tTG cDNA in antisense orientation resulted in a pronounced decrease of both spontaneous and retinoic acid (RA)-induced apoptosis. We also present evidence that (i) the apoptotic program of these neuroectodermal cells is strictly regulated by RA and (ii) cell death by apoptosis in the human neuroblastoma SK-N-BE(2) cells preferentially occurs in the substrate-adherent phenotype. For the first time, we report here a direct effect of tTG in the phenotypic maturation toward apoptosis. These results indicate that the tTG-dependent irreversible cross-linking of intracellular protein represents an important biochemical event in the induction of the structural changes featuring cells dying by apoptosis.     

Inhibition of "tissue" transglutaminase increases cell survival by preventing apoptosis

Treatment of the human promonocytic cell line U937 with all-trans-retinoic acid (RA) commits these cells to apoptosis, which can be triggered by simply increasing intracellular calcium levels by the ionophore A23187. RA treatment of U937 cells is characterized by a decrease in Bcl-2 and marked induction of "tissue" transglutaminase (tTG) gene expression. In this study, we show that the inhibition of tTG expression in U937 cells undergoing apoptosis prevents their death. In fact, U937 cell-derived clones transfected with the human tTG gene in the antisense orientation showed a pronounced decrease in apoptosis induced by several stimuli. These findings demonstrate that the Ca(2+)-dependent irreversible cross-linking of intracellular proteins catalyzed by tTG represents an important biochemical event in the gene-regulated cell death in monoblasts. In addition, our data indicate that the apoptotic program in promonocytic cells is strictly regulated by RA and that a key role is played by the free intracellular calcium concentration.     

The expression of "tissue" transglutaminase in two human cancer cell lines is related with the programmed cell death (apoptosis).

The expression of "tissue" transglutaminase (tTG) in two human tumor cell lines (the cervix adenocarcinoma line HeLa-TV and the neuroblastoma cells SK-N-BE-2) was found to be in correlation with the rate of physiological cell death (apoptosis) in culture. We investigated the effect of retinoic acid (RA) and alpha-difluoromethylornithine (DFMO) in order to elucidate the relationship between tTG expression and apoptosis. RA led to a 6-fold increase of tTG activity in HeLa-TV cells and to a 12-fold increase in SK-N-BE(2) cells, which was paralleled in both cell lines by a proportional increase in the number of apoptotic bodies recovered from the cultures. On the contrary, DFMO determined a dramatic reduction of tTG expression and of the apoptotic index. Immunohistochemical analysis using an anti-tTG antibody showed that the enzyme was accumulated in both cell lines within typical apoptotic bodies. Immunocytochemistry and cell cloning of SK-N-BE(2) line demonstrated that tTG was absent in cells showing neurite outgrowth, indicating that the enzyme expression is not associated with neural differentiation, even though both phenomena are elicited by retinoic acid. On the whole, these data indicate that also in tumors tTG activation takes place in cells undergoing apoptosis. The enzyme is activated in apoptotic cells to form cross-linked protein envelopes which are insoluble in detergents and chaotropic agents. The number of insoluble protein envelopes as well as the N,N-bis(gamma-glutamyl)polyamine cross-links is related with both tTG expression and apoptotic index, strongly suggesting the participation of the enzyme in the apoptotic program.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation of tissue transglutaminase in rat hepatoma cell lines McA-RH7777 and McA-RH8994: Relation to growth rate and cell death

Close correlation between tissue transglutaminase (tTG) induction and growth regulation and/or cell death processes has been suggested in many cell lineages. In this study, the regulation of the tTG levels by various growth and differentiation factors and its relation to growth rate and cell death processes were investigated in two rat hepatoma cell lines, McA-RH7777 and McA-RH8994, using a monoclonal antibody against liver tTG. Transforming growth factor-β1 (TGF-β1) and retinoic acid (RA) each increased tTG to the level of 8- to 32-fold above that of control cultures in both cell lines after 72-h treatment. Dexamethasone (DEX) induced a 16- to 32-fold of tTG in McA-RH8994 cells while it did not change the enzyme level in McA-RH7777 cells. Simultaneous addition of DEX and RA increased the tTG level to more than 50-fold in McA-RH7777 cells as well as McA-RH8994 cells. Other factors, such as TGF-α, hepatocyte growth factor, dimethyl sulfoxide, and protein kinase C activator, did not show significant increases of the tTG levels. Although tTG induction by TGF-β1 or DEX appeared to be correlated with their growth suppressive effects, RA increased the tTG level without suppressing the growth rate of hepatoma cells. TGF-β1 was also shown to induce cell death in both cell lines. Our results demonstrate that RA and DEX are capable of modulating the TGF-β1-induced cell death processes independent of the tTG levels. We present evidence here that tTG induction by itself is not the direct cause of growth suppression and cell death in these hepatoma cells.     

Processes involved in retinoic acid production of small embryonic palatal shelves and limb defects

All-trans-retinoic acid (RA) is teratogenic to the embryonic mouse, producing malformations in many developing systems, including the limb bud and palate. High incidences of limb defects and cleft palate are induced at doses which are not maternally toxic and do not increase resorptions. Exposure to RA on gestational day (GD) 10 results in small palatal shelves, which fail to make contact on GD 14. The formation of small shelves could be a consequence of increased cell death, reduced proliferation, a combination of these effects, or some other effect such as inhibition of extracellular matrix production. After exposure to 100 mg RA/kg on GD 10, proliferation in mesenchymal cells of the palatal shelves was not reduced from GD 12 to GD 14 and the levels of cell death in control and treated shelves did not differ when observed by light and electron microscopy. The present study examines the effects of RA on cell death and proliferation from GDs 10-12 and compares the effects in palatal shelves and limb buds. Embryonic mice were exposed to RA suspended in corn oil (100 mg/kg on GD 10), a dose that was teratogenic but not maternally toxic or embryolethal. Embryos were collected at 4, 12, 24, 36, or 48 hr postexposure, and tissues which form the palate or limb were dissected from the embryos, stained by a modified Feulgen procedure, and whole mounted on slides. Mitotic index (MI) and percentage dead cells were determined for mesenchymal cells of the first visceral arch, maxillary process, or palatal shelf (depending on stage of development) and forelimb buds. In the palatal tissues from GD 10 to GD 12, RA did not significantly alter MI and percentage dead cells was significantly increased only at 4 hr postexposure. Some whole embryos were prepared for scanning electron microscopy (SEM). At 48 hr (GD 12) a reduction in the size of the shelves was not apparent on SEM. In the limb buds, RA did not increase percentage dead cells, but MI was significantly decreased. A decreasing rate of proliferation was detected in control facial tissues as development progressed, and this agrees with findings in rat and chick. Thus it appears that mesenchymal cell death and reduced proliferation are not responsible for the small palatal shelves seen on GD 14. RA did not increase cell death but inhibited proliferation in the limb bud, and this effect may contribute to the retarded development and malformations occurring in the limb.     

Induction of apoptosis by all-trans retinoic acid in the human myeloma cell line RPMI 8226 and negative regulation of some of its typical morphological features by dexamethasone.

We investigated the effects of all-trans retinoic acid (RA) and dexamethasone (Dex) on the in vitro growth of the human myeloma cell line RPMI 8226. RA inhibited RPMI 8226 cell growth by both antiproliferative effect and induction of apoptosis. Typical morphological and biochemical characteristics of apoptosis including chromatin condensation, apoptotic bodies formation and internucleosomal DNA cleavage were detected after 4 days of treatment with 1 microM RA. In situ TUNEL assay demonstrated that DNA cleavage preceded chromatin condensation. The expression of tissue transglutaminase (tTG), an enzyme proposed to play a role in apoptosis was induced with RA, as shown by both enzymatic assay and in situ immunofluorescence detection. Dex, when used alone, had no effect on cell growth and apoptosis. When combined to RA, Dex did not interfere with the RA-dependent inhibition of cell proliferation, but unexpectedly inhibited both quantitatively and qualitatively several morphological and biochemical features of the apoptosis induced by RA. Dex did not affect RA-induced DNA breaks formation but impeded the progression of chromatin condensation and the formation of apoptotic bodies. Interestingly, Dex also inhibited the RA-dependent induction of tTG. RU486, a glucocorticoid antagonist, counteracted all Dex effects. Taken together these data demonstrate that key cytoplasmic and nuclear events occurring during apoptosis are differentially regulated by RA and Dex in myeloma cell line RPMI 8226.     

Patterning of forelimb bud myogenic precursor cells requires retinoic acid signaling initiated by Raldh2

Limb skeletal muscle is derived from cells of the dermomyotome that detach and migrate into the limb buds to form separate dorsal and ventral myogenic precursor domains. Myogenic precursor cell migration is dependent on limb bud mesenchymal expression of hepatocyte growth factor/scatter factor (Hgf), which encodes a secreted ligand that signals to dermomyotome through the membrane receptor tyrosine kinase Met. Here, we find that correct patterning of Hgf expression in forelimb buds is dependent on retinoic acid (RA) synthesized by retinaldehyde dehydrogenase 2 (Raldh2) expressed proximally. Raldh2(-/-) forelimb buds lack RA and display an anteroproximal shift in expression of Hgf such that its normally separate dorsal and ventral expression domains are joined into a single anterior-proximal domain. Met and MyoD are expressed in this abnormal domain, indicating that myogenic cell migration and differentiation are occurring in the absence of RA, but in an abnormal location. An RA-reporter transgene revealed that RA signaling in the forelimb bud normally exists in a gradient across the proximodistal axis, but uniformly across the anteroposterior axis, with all proximal limb bud cells exhibiting activity. Expression of Bmp4, an inhibitor of Hgf expression, is increased and shifted anteroproximally in Raldh2(-/-) limb buds, thus encroaching into the normal expression domain of Hgf. Our studies suggest that RA signaling provides proximodistal information for limb buds that counterbalances Bmp signaling, which in turn helps mediate proximodistal and anteroposterior patterning of Hgf expression to correctly direct migration of Met-expressing myogenic precursor cells.     

"Chemical surgery" as an approach to study morphogenetic events in embryonic mouse limb

Cytosine arabinoside (Ara-C) or retinoic acid (RA) was injected into pregnant mice in doses which induce a high incidence of limb defects. Within 4 hr of the treatment, extensive cell death was observed in the embryonic limb buds. However, the location of necrotic cells and the eventual limb defects were different for the two chemicals. Ara-C killed cells in those regions of the limb which were undergoing active proliferation. RA, on the other hand, had no effect on actively dividing cells but was lethal to cells of chondrogenic lineage at stages when their proliferation rate had fallen 7- to 10-fold below the original rate. In all cases, an excellent correlation between the location of dead cells (as seen 4 hr after drug treatment) and the eventual bony defects (as seen in the term fetuses) was observed. The unique properties of Ara-C and RA have been exploited in determining the relative levels of cytodifferentiation in the embryonic mouse limb buds. It is concluded that in the limbs of early 11th day mouse embryos (comparable to chick stage 19–20), differentiation of future skeletal elements has not yet begun. However, by the 12th day (comparable to chick stage 23), cell populations destined to form most of the future cartilages (except for digits) have already been established.     

Autoregulation of Shh expression and Shh induction of cell death suggest a mechanism for modulating polarising activity during chick limb development

The polarising region expresses the signalling molecule sonic hedgehog (Shh), and is an embryonic signalling centre essential for outgrowth and patterning of the vertebrate limb. Previous work has suggested that there is a buffering mechanism that regulates polarising activity. Little is known about how the number of Shh-expressing cells is controlled but, paradoxically, the polarising region appears to overlap with the posterior necrotic zone, a region of programmed cell death. We have investigated how Shh expression and cell death respond when levels of polarising activity are altered, and show an autoregulatory effect of Shh on Shh expression and that Shh affects cell death in the posterior necrotic zone. When we increased Shh signalling, by grafting polarising region cells or applying Shh protein beads, this led to a reduction in the endogenous Shh domain and an increase in posterior cell death. In contrast, cells in other necrotic regions of the limb bud, including the interdigital areas, were rescued from death by Shh protein. Application of Shh protein to late limb buds also caused alterations in digit morphogenesis. When we reduced the number of Shh-expressing cells in the polarising region by surgery or drug-induced killing, this led to an expansion of the Shh domain and a decrease in the number of dead cells. Furthermore, direct prevention of cell death using a retroviral vector expressing Bcl2 led to an increase in Shh expression. Finally, we provide evidence that the fate of some of the Shh-expressing cells in the polarising region is to undergo apoptosis and contribute to the posterior necrotic zone during normal limb development. Taken together, these results show that there is a buffering system that regulates the number of Shh-expressing cells and thus polarising activity during limb development. They also suggest that cell death induced by Shh could be the cellular mechanism involved. Such an autoregulatory process based on cell death could represent a general way for regulating patterning signals in embryos.     

BCL-2 protects peroxynitrite-treated thymocytes from poly(ADP-ribose) synthase (PARS)-independent apoptotic but not from PARS-mediated necrotic cell death

In thymocytes, peroxynitrite induces poly(ADP-ribose) synthetase (PARS) activation, which results in necrotic cell death. In the absence of PARS, however, peroxynitrite-treated thymocytes die by apoptosis. Because Bcl-2 has been reported to inhibit not only apoptotic but also some forms of necrotic cell death, here we have investigated how Bcl-2 regulates the peroxynitrite-induced apoptotic and necrotic cell death. We have found that Bcl-2 did not provide protection against peroxynitrite-induced necrotic death, as characterized by propidium iodide uptake, mitochondrial membrane potential decrease, secondary superoxide production, and cardiolipin loss. In the presence of a PARS inhibitor, peroxynitrite-treated thymocytes from Bcl-2 transgenic mice showed no caspase activation or DNA fragmentation and displayed smaller mitochondrial membrane potential decrease. These data show that Bcl-2 protects thymocytes from peroxynitrite-induced apoptosis at a step proximal to mitochondrial alterations but fails to prevent PARS-mediated necrotic cell death. Activation of tissue transglutaminase (tTG) occurs in various forms of apoptosis. Peroxynitrite did not induce transglutaminase activity in thymocytes and did not have a direct inhibitory effect on the purified tTG. Basal tTG was not different in Bcl-2 transgenic and wild type cells.     

Tissue transglutaminase-dependent posttranslational modification of the retinoblastoma gene product in promonocytic cells undergoing apoptosis.

The retinoblastoma gene product (pRB) plays an important role in controlling both cell release from the G1 phase and apoptosis. We show here that in the early phases of apoptosis, pRB is posttranslationally modified by a tissue transglutaminase (tTG)-catalyzed reaction. In fact, by employing a novel haptenized lysis synthetic substrate which allows the isolation of glutaminyl-tTG substrates in vivo, we identified pRB as a potential tTG substrate in U937 cells undergoing apoptosis. In keeping with this finding, we showed that apoptosis of U937 cells is characterized by the rapid disappearance of the 105,000- to 110,000-molecular-weight pRB forms concomitantly with the appearance of a smear of immunoreactive products with a molecular weight of greater than 250,000. The shift in pRB molecular weight was reproduced by adding exogenous purified tTG to extracts obtained from viable U937 cells and was prevented by dansylcadaverine, a potent enzyme inhibitor. The effect of the pRB posttranslational modification during apoptosis was investigated by determining the E2F-1 levels and by isolating and characterizing pRB-null clones from U937 cells. Notably, the lack of pRB in these U937-derived clones renders these p53-null cells highly resistant to apoptosis induced by serum withdrawal, calphostin C, and ceramide. Taken together, these data suggest that tTG, acting on the pRB protein, might play an important role in the cell progression through the death program.     

Essential roles of retinoic acid signaling in interdigital apoptosis and control of BMP-7 expression in mouse autopods

We previously reported that mice lacking the RARgamma gene and one or both alleles of the RARbeta gene (i.e., RARbeta+/-/RARgamma-/- and RARbeta-/-/RARgamma-/- mutants) display a severe and fully penetrant interdigital webbing (soft tissue syndactyly), caused by the persistence of the fetal interdigital mesenchyme (Ghyselinck et al., 1997, Int. J. Dev. Biol. 41, 425-447). In the present study, these compound mutants were used to investigate the cellular and molecular mechanisms involved in retinoic acid (RA)-dependent formation of the interdigital necrotic zones (INZs). The mutant INZs show a marked decrease in the number of apoptotic cells accompanied by an increase of cell proliferation. This marked decrease was not paralleled by a reduction of the number of macrophages, indicating that the chemotactic cues which normally attract these cells into the INZs were not affected. The expression of a number of genes known to be involved in the establishment of the INZs, the patterning of the autopod, and/or the initiation of apoptosis was also unaffected. These genes included BMP-2, BMP-4, Msx-1, Msx-2, 5' members of Hox complexes, Bcl2, Bax, and p53. In contrast, the mutant INZs displayed a specific, graded, down-regulation of tissue transglutaminase (tTG) promoter activity and of stromelysin-3 expression upon the removal of one or both alleles of the RARbeta gene from the RARgamma null genetic background. As retinoic acid response elements are present in the promoter regions of both tTG and stromelysin-3 genes, we propose that RA might increase the amount of cell death in the INZs through a direct modulation of tTG expression and that it also contributes to the process of tissue remodeling, which accompanies cell death, through an up-regulation of stromelysin-3 expression in the INZs. Approximately 10% of the RARbeta-/- /RARgamma-/- mutants displayed a supernumerary preaxial digit on hindfeet, which is also a feature of the BMP-7 null phenotype (Dudley et al., 1995, Genes Dev. 9, 2795-2807; Luo et al., 1995, Genes Dev. 9, 2808-2820). BMP-7 was globally down-regulated at an early stage in the autopods of these RAR double null mutants, prior to the appearance of the digital rays. Therefore, RA may exert some of its effects on anteroposterior autopod patterning through controlling BMP-7 expression.     

Tissue transglutaminase triggers oligomerization and activation of dual leucine zipper-bearing kinase in calphostin C-treated cells to facilitate apoptosis

Although tissue transglutaminase (tTG) has been recognized as a mediator of apoptosis in various experimental models, little is currently known about the molecular mechanisms by which this protein modulates cell death. Recent work from our laboratory has shown that activation of tTG in cells exposed to the apoptotic inducer calphostin C triggers the crosslinking of dual leucine zipper-bearing kinase (DLK), a proapoptotic kinase acting as an essential component of the c-Jun amino-terminal kinase (JNK) signaling pathway. As a consequence of this observation, we have undertaken experiments to investigate the functional relevance of DLK oligomerization in tTG-mediated apoptosis. Our results indicate that, in cells undergoing calphostin C-induced apoptosis, tTG-dependent DLK oligomerization occurs early in the apoptotic response. Both immunocomplex kinase assays and immunoblotting with phosphospecific antibodies revealed that oligomer formation by tTG-mediated crosslinking reactions significantly enhanced the kinase activity of DLK and its ability to activate the JNK pathway. Moreover, functional studies demonstrate that tTG-mediated oligomerization of wild-type DLK sensitizes cells to calphostin C-induced apoptosis, while crosslinking of a kinase-inactive variant of DLK does not. Collectively, these data strongly suggest that tTG facilitates apoptosis, at least partly, by oligomerization and activation of the proapoptotic kinase DLK.     

Intracellular localization and activity state of tissue transglutaminase differentially impacts cell death

Tissue transglutaminase (tTG) is a unique member of the transglutaminase family as it is both a transamidating enzyme and a GTPase. In the cell tTG is mostly cytosolic, however it is also found in the nucleus and associated with the plasma membrane. tTG can be proapoptotic, however anti-apoptotic activities of the enzyme have also been reported. To determine how the intracellular localization and transamidating activity of tTG modulates its effects on apoptosis, HEK293 cells were transiently transfected with tTG or [C277S]tTG (which lacks transamidating activity) constructs that were targeted to different intracellular compartments. Apoptosis was induced by thapsigargin treatment, which results in increased intracellular calcium concentrations. Cytosolic tTG was pro-apoptotic, while nuclear localization of [C277S]tTG attenuated apoptosis. Membrane-targeted tTG had neither pro- nor anti-apoptotic functions. This finding indicates for the first time that intracellular localization is an important determinant of the effect of tTG on apoptosis. Previous studies have suggested that tTG may modulate retinoblastoma (Rb) protein, an important suppressor of apoptosis. tTG interacted with Rb and after induction of apoptosis, the interaction of nuclear-targeted [C277S]tTG with Rb was increased significantly concomitant with an attenuation of apoptosis. In contrast, the interaction of nuclear-targeted tTG with Rb was significantly decreased and apoptosis was not attenuated. These data suggest that tTG protects cells against apoptosis in response to stimuli that do not result in increased transamidating activity by translocating to the nucleus, and that complexing with Rb may be an important aspect of the protective effects of tTG.     

Retinoic acid respecifies limb bud cells in vitro.

Retinoic acid (RA) is known to have dramatic effects on limb pattern formation and has been shown to exert its effects on limbs by converting anterior limb bud cells into cells with posterior positional properties. In this study we find that dissociated posterior limb bud cells from chick and mouse embryos cultured at high density (micromass cultures) are able to stimulate the formation of supernumerary digits when grafted into developing wing buds and that the positional identity of both chick and mouse limb bud cells can be maintained for finite periods of time in vitro. Furthermore, using this assay system we have tested whether anterior cells from mouse and chick limb buds can be converted into cells with posterior identity by exposure to RA in vitro. We find that anterior limb bud cells acquire posterior properties after culture in the presence of RA.     

Effects of tissue transglutaminase on retinoic acid-induced cellular differentiation and protection against apoptosis.

Retinoic acid (RA) and its various synthetic analogs affect mammalian cell growth, differentiation, and apoptosis. Whereas treatment of the human leukemia cell line HL60 with RA results in cellular differentiation, addition of the synthetic retinoid, N-(4-hydroxyphenyl) retinamide (HPR), induces HL60 cells to undergo apoptosis. Moreover, pretreatment of HL60 cells as well as other cell lines (i.e. NIH3T3 cells) with RA blocks HPR-induced cell death. In attempting to discover the underlying biochemical activities that might account for these cellular effects, we found that monodansylcadaverine (MDC), which binds to the enzyme (transamidase) active site of tissue transglutaminase (TGase), eliminated RA protection against cell death and in fact caused RA to become an apoptotic factor, suggesting that the ability of RA to protect against apoptosis is linked to the expression of active TGase. Furthermore, it was determined that expression of exogenous TGase in cells exhibited enhanced GTP binding and transamidation activities and mimicked the survival advantage imparted by RA. We tested whether the ability of this dual function enzyme to limit HPR-mediated apoptosis was a result of the ability of TGase to bind GTP and/or catalyze transamidation and found that GTP binding was sufficient for the protective effect. Moreover, excessive transamidation activity did not appear to be detrimental to cell viability. These findings, taken together with observations that the TGase is frequently up-regulated by environmental stresses, suggest that TGase may function to ensure cell survival under conditions of differentiation and cell stress.     

3-Deazauridine triggers dose-dependent apoptosis in myeloid leukemia cells and enhances retinoic acid-induced granulocytic differentiation of HL-60 cells

Therapeutic nucleoside analogue 3-deazauridine (DU) exerts cytotoxic activity against cancer cells by disruption of DNA synthesis resulting in cell death. The present study evaluates whether DU alone at doses 2.5-15 microM or in combination with all trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) is effective against myelogenous leukemia. The data of this study indicate that DU induces dose-dependent cell death by apoptosis in myeloid leukemia cell lines HL-60, NB4, HEL and K562 as demonstrated by cell staining or flow cytometry and agarose gel electrophoresis. 24h-treatment with DU produced dose-dependent HL-60 cell growth inhibition and dose-independent S phase arrest that was not reversed upon removal of higher doses of DU (10-15 microM). Exposition to nontoxic dose of DU (2.5 microM) for 24h followed by RA or dbcAMP and 96 h-cotreatment with DU significantly enhanced RA- but not dbcAMP-mediated granulocytic differentiation. Cell maturation was paralleled with an increase in the proportion of cells in G1 or G2+M phase. We conclude that, depending on the dose or the sequence of administration with RA, an inhibitor of DNA replication, DU triggers a process of either differentiation or apoptosis in myeloid leukemia cells.     

Spatial and temporal patterns of cell death in limb bud mesoderm after apical ectodermal ridge removal

A spatiotemporal pattern of cell death occurred in the chick wing and leg bud mesoderm after removal of apical ectodermal ridge at stages 18–20. Cells died in a region extending from the limb bud distal surface to 150–200 μm into the mesoderm. Limb buds from which ridge was removed at later stages in development did not exhibit a spatiotemporal pattern of cell death. In control experiments in which dorsal ectoderm was removed, a pattern of cell death did not occur. Removal of the ridge and part of the 150- to 200-μm zone of prospective cell death resulted in cell death in an area approximately equal to the amount of the zone remaining. After removal of all of the prospective zone of cell death plus the apical ridge, cell death was observed in the remaining limb bud mesoderm. In these limb buds, cell death occurred in a region in which it had not been seen in limb bud with apical ridge alone removed. We conclude that at stages 18–20 the mesodermal cells 150–200 μm beneath the ridge require the apical ridge to survive. More proximal mesodermal cells do not die after ridge removal alone, but apparently require the presence of the more distal mesoderm to survive. Whether this is a requirement for something intrinsic to the distal mesoderm or something it possesses by way of the ridge is unknown. After stage 23, the limb mesoderm cells do not die when the apical ridge is removed. Nevertheless, at the later stages, ridge continues to be required for limb bud proximal-distal elongation and the differentiation of distal limb elements.