REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS : II. Induction of DNA Synthesis and Cell Division by Isoleucine and Glutamine in G1-Arrested Cells in Suspension Culture

Suspension cultures of Chinese hamster cells (line CHO), which had stopped dividing and were arrested in G₁ following growth to high cell concentrations in F-10 medium, could be induced to reinitiate DNA synthesis and to divide in synchrony upon addition of the appropriate amounts of isoleucine and glutamine. Both amino acids were required to initiate resumption of cell-cycle traverse. Deficiencies in other amino acids contained in F-10 medium did not result in accumulation of cells in G₁, indicating a specific action produced by limiting quantities of isoleucine and glutamine. In the presence of sufficient glutamine, approximately 2 x 10^-6 M isoleucine was required for all cells to initiate DNA synthesis in a population initially containing 1.5 x 10⁵ cells/ml. Under similar conditions, about 4 x 10^-6 M isoleucine was required for all G₁-arrested cells to progress through cell division. In contrast, 1 x 10^-4 M glutamine was necessary for maximum initiation of DNA synthesis in G₁ cells, along with sufficient isoleucine. A technique for rapid production of G₁-arrested cells is described in which cells from an exponentially growing population placed in F-10 medium deficient in both isoleucine and glutamine or isoleucine alone accumulated in G₁ after 30 hr.     

Ganglioside GM1 metabolism in living human fibroblasts with β-galactosidase deficiency

Summary The uptake and catabolism of [³H-ceramide]-G_M1 was followed in living fibroblasts from patient with different forms of -galactosidase deficiency. Gangliosides are identified according to the nomenclature of Svennerholm (1963). A total inability to metabolize the ingested substrate was found in infantile G_M1-gangliosidosis whereas cells from an adult G_M1-gangliosidosis variant showed a slower rate of degradation, compared with controls. Morquio B fibroblasts had a comparable catabolism of G_M1 as controls. Fibroblasts from different types of galactosialidosis, a recessive disease associated with a coexistent -galactosidase/neuraminidase deficiency all showed degradation of ingested G_M1. In view of the molecular defect in this disease, this catabolism must be due to the 10–20% of monomeric -galactosidase molecules present in the lysosomes. Unexpectedly, in these cells an impaired metabolism of G_M3 was found. The same finding was observed when cells with an isolated neuraminidase deficiency (mucolipidosis I) were loated with G_M1. A hypothesis is presented to explain these results.     

Fractionation and specificity of DNA methylases from the Escherichia coli SK cells

Summary Specificity of DNA methylation enzymes from the E. coli SK cells and conditions for their separation have been investigated. Column chromatography on carboxymethylcellulose permits fractionation of methylase activity into six discrete peaks whose specificity to the methylated base has been determined in vitro with H³-SAM as precursor. All methylases specific for adenine produced 6-methylaminopurine, all methylases specific for cytosine yielded 5-methylcytosine.The first enzymatic activity peak containing cytosine methylase free of traces of adenine-methyiating activity (E₁), and the second peak containing both the enzymes (E₂) were not adsorbed on the ion exchanger and went off the column with the effluent (column buffer). Adenine specific methylase E₂ is retarded to a small extent during the passage through the column. The second adenine methylases (W) was characterized by weak bonds with the ion exchanger and was removed when washing the column with column buffer. The elution with NaCl gradient produced successively three enzymatic activity peaks: adenine methylase (G_I), cytosine methylase (G_II), and one more adenine methylase (G_III) removed from the column by 0.16 m, 0.24 m and 0.43 m NaCl respectively.Using a new modification of the complementary methylation test, the specificity with regard to recognition site was examined for all enzymes, except for W and G_III, which were extremely unstable. The adenine methylases E₂ and G_I and the cytosine methylases E₁ and G_II were shown to recognize different sites and to be different enzymes. In view of the drastic differences in their chromatographic behaviour and physical stability, the adenine methylases W and G_III are probably also different enzymes.     

GM1-gangliosidosis: Accumulation of ganglioside GM1 in cultured skin fibroblasts and correlation with clinical types

Summary Uptake of radioactivity from ¹⁴C-galactose into gangliosides by cultured skin fibroblasts was studied. G_M3 was the major ganglioside in control human fibroblasts. An increase of G_M1 was demonstrated in G_M1-gangliosidosis fibroblasts. The degree of G_M1 accumulation was correlated with the clinical types of this disease. The fibroblasts from an infantile-type patient showed a marked increase of G_M1. In late-onset types the amount of total gangliosides was only slightly increased, but the distribution of individual gangliosides was definitely abnormal; a relative increase of G_M1 was demonstrated in these cases. G_M1 -galactosidase activities were not detectable in either infantile or late-onset cases.     

Location of heitz's zerstäubungsstadium (dispersion phase) in the mitotic cycle ofPhaseolus coccineus and the concept of Angiosperm endomitosis

Summary DNA microdensitometry and autoradiography after treatment with³H-thymidine were used to study the phase of dispersion of chromocenters (Z phase) in parallel with chromocentric nuclei inPhaseolus coccineus. In all materials studied, two types of chromocentric nuclei were present.In radicle apices of dry seeds, two classes of nuclear DNA contents were measured, 2 C (G₁) and 4 C (G₂). The 2 C DNA class comprised all chromocentric type I nuclei, the 4 C class included Z phases and chromocentric type II nuclei. The 4 C (G₂) condition of Z phases implies that Z phases maintain their nuclear structure for some time after the end of DNA replication. Shoot apices also contain 2 C (G₁) and 4 C (G₂) nuclei but 4 C nuclei (Z phases and chromocentric type II nuclei) are rare.In seedling root apices, Z phases are from 1.02 to 4.08 times as frequent as prophases. This excludes that Z phase is a very early prophase. DNA microdensitometry shows that the chromocentric type I includes 2 C (G₁) nuclei and nuclei in the first part of the S phase, Z phases include 4 C (G₂) nuclei and nuclei in the last stage of the S phase and chromocentric type II includes mainly 4 C (G₂) nuclei and nuclei in the second part of S. After 90 minutes of treatment with³H-thymidine all Z phase nuclei are labeled. This result and the microdensitometric data unequivocally demonstrate that Z phase is located at the end of S.The present results and those of previous authors on Z phase are discussed in relation to Geitler's concept of Angiosperm endomitosis. It is concluded that the term Angiosperm endomitosis must be abandoned and substituted by the term chromosome endoreduplication.     

Monoclonal antibodies specific to plant tubulin

Summary Tubulin was isolated from mung bean seedling by a combination of affinity (ethyl N-phenylcarbamate-Sepharose 4 B) and ion exchange (DEAE-Sephacel) chromatography. Using SDS-PAGE together with blotting with subunit-specific antitubulins, mung bean tubulin has been shown to consist of two -tubulin subunits, MBT₂ and MBT₃, of which MBT₃ is a minor component, and one -tubulin, MBT₁.Monoclonal antibodies were produced by fusing mouse myeloma cells and spleen cells from a Balb/c mouse immunized with mung bean tubulin. Antibody producing cell lines were identified by an ELISA assay and immunofluorescence microscopy and subsequently cloned by limiting dilution.The properties of monoclonal antibody (K₄E₇G₃) were examined by Western blot analysis and indirect immunofluorescence studies. K₄E₇G₃ reacts with MBT₂ and MBT₃ -tubulin subunits of mung bean tubulin, but not with MBT₁ -tubulin nor with the - and -subunits of sheep brain tubulin. Peptide fragments transferred onto nitrocellulose papers were treated with K₄E₇G₃ and with other monoclonal antibodies that are known to be specific to the -subunit of yeast tubulin and - or -subunit of mammalian brain tubulin. MBT₂ and MBT₃ are shown to be similar but not identical and are quite different from MBT₁ and the -subunit of sheep brain tubulin. K₄E₇G₃ reacts with peptide fragments in MBT₂ and MBT₃ that are not found in digests of brain tubulin, and that are either not reactive or only weakly reactive to the antibodies to yeast and brain -tubulin. It is concluded that K₄E₇G₃ and another monoclonal antibody, K₂D₇B₈, which has similar properties, are relatively specific for plant -tubulin.In indirect immunofluorescence studies on a wide range of plant cells, the epitopes recognised by these monoclonal antibodies are shown to be present in all types of microtubule array that were investigated. The spindle, preprophase band, phragmoplast and interphase microtubules were clearly observed in onion and mung bean root tip cells. Reactions with spindle microtubules ofFunaria spore mother cells and with the blepharoplast and flagella microtubules of fern spermatozoa are also seen. However, studies using several animal cell lines have shown that K₄E₇G₃ and K₂D₇B₈ do not give positive immunofluorescent localization of animal microtubules, correlating with the inability of K₄E₇G₃ to react with brain tubulin subunits on Western blot analysis.     

Effects of growth temperature upshift on cell cycle progression in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cell cycle progression of Cryptococcus neoformans was studied for cells grown exponentially at 15°, 24°, and 30°C. Except for speed, cell cycle progression was similar. In particular, budding occurred relatively soon after initiation of DNA synthesis at 15°, 24°, and 30°C. After growth temperature was shifted from 15° to 30°C, cells were transiently arrested before initiation of DNA synthesis. Thus, similar to Saccharomyces erevisiae, Start was the main susceptible cell cycle controlling point in C. neoformans. However, after spontaneous release from arrest as above, cells were further arrested in the unbudded state. Thus, the timing of budding was delayed just before the G₂ phase, or even until after entering the G₂ phase, but it was also transient, and 5h after the shift buds emerged relatively soon after initiation of DNA synthesis. Thus, C. neoformans cells can respond adaptively to mild stress by delaying budding. The existence of the second susceptible cell cycle control point, i.e., budding, appears to endow C. neoformans with a unique characteristic of stronger inhibition of multiplication than growth. A model of the C. neoformans cell cycle is also presented.     

Structural studies of gangliosides from the YAC-1 mouse lymphoma cell line by immunological detection and fast atom bombardment mass spectrometry

YAC-1 cells were propagated in bioreactors in 11 and 7.51 volumes. The cells were metabolically labelled withd-[1-¹⁴C]galactose andd-[1-¹⁴C]glucosamine. The ganglioside fraction, purified by DEAE-Sepharose and silica gel column chromatography, showed on thin layer chromatography four major bands with mobilities between G_M1 and G_D1a. Gangliosides, obtained by further purification steps including high performance liquid chromatography on silica gel 60 columns with a gradient system of isopropanol:hexane:water, and preparative high performance TLC were characterized by (1) immunostaining of corresponding asialogangliosides obtained by mild acid hydrolysis and neuraminidase treatment and (2) fast atom bombardment mass spectrometry of native and permethylated samples and methylation analysis of G_M1b ganglioside. As well as small amounts of G_M2 and G_M1, the major gangliosides found in the complex mixture were G_M1b and GalNAc-G_M1b. The structural heterogeneity of these gangliosides was cased by (a) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C_16:0, C_24:0, C_24:1) and (b) N-substitution of the sialic acid moieties with either acetyl or glycolyl groups. Disialogangliosides were detected only in low amounts and will be the subject of further investigation. A polyclonal chicken antiserum was raised against IVNeuAc-GgOse₅Cer. The antiserum was highly specific for gangliosides (IVNeuAc and IVNeuGc) and asialogangliosides with a GgOse₅Cer backbone. No cross-reaction with G_M1b or GgOse₄Cer was observed. Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography, HPTLC, high performance thin layer chromatography; NK, natural killer; SIM, selective ion monitoring; TIC, total ion current. NeuAc,N-acetylneuraminic acid; NeuGc,N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUB-IUPAC recommendations. GgOse₃Cer or gangliotriaosylceramide or asialo G_M2, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide or asialo G_M1, Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₅Cer organgliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; II³NeuAc-GgOse₄Cer or G_M1; IV³NeuAcGgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³(NeuAc)₂-GgOse₄Cer or G_D1c; IV³NeuAc,III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc,II³(NeuAc)₂-GgOse₄Cer or G_T1b;Vibrio cholerae and Arthrobacter ureafaciens neuraminidase (EC 3.2.1.18).     

Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G2/M Checkpoint Arrest

ATM-dependent initiation of the radiation-induced G₂/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G₂ phase are repaired by DNA nonhomologous end joining (NHEJ), while ∼15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G₂/M checkpoint is maintained in irradiated G₂ cells, in light of our current understanding of G₂ phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1^−/− and MDC1^−/− cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.DNA double-strand breaks (DSBs) activate the DNA damage response (DDR), a coordinated process that functions to enhance survival and maintain genomic stability. The DDR includes pathways of DSB repair and a signal transduction response that activates apoptosis and cell cycle checkpoint arrest and influences DSB repair (15). DNA nonhomologous end joining (NHEJ) and homologous recombination (HR) represent the major DSB repair mechanisms, NHEJ being the major mechanism in G₀/G₁, while both processes function in G₂ (9, 32). Ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) are related phosphoinositol 3-kinase-like kinases (PIKKs) that regulate the DNA damage signaling response. ATM is activated by DSBs, while ATR is activated at single-strand (ss) regions of DNA via a process that involves ATRIP-replication protein A (RPA)-ssDNA association. Ionizing radiation (IR) induces DSBs, base damage, and ss nicks. Since neither base damage nor ss nicks activate ATR, IR-induced signaling in the G₁ and G₂ phases is predominantly ATM dependent (3, 29). In S phase, ATR can be activated by both endogenous and exogenously induced lesions following replication fork stalling/collapse (8).Recent work has shown that in G₂ phase, DSBs can undergo resection via an ATM-dependent process generating ssDNA regions that can activate ATR following RPA association (11). ATR activation at resected DSBs is coupled to loss of ATM activation (11). Although ATM and ATR share overlapping substrates, there is specificity in their signaling to the transducer kinases; ATM uniquely phosphorylates Chk2, while ATR phosphorylates Chk1. Phosphorylation of either Chk1 or Chk2 causes their activation. Critical targets of Chk1/Chk2 are the Cdc25 phosphatases, which regulate the cyclin-dependent kinases (Cdks), including Cdk1, the regulator of mitotic entry (18). Collectively, these studies suggest that two components of ATM-dependent signaling to the G₂/M checkpoint machinery can occur: ATM-Chk2 signaling at unresected DSBs and ATM-ATR-Chk1 signaling at resected DSBs.Although much is known about the mechanism leading to G₂/M checkpoint activation, few studies have addressed how arrest is maintained and how release coordinates with the status of DSB repair. We examine here the maintenance of checkpoint arrest during the immediate phase of DSB repair. We do not address the issue of checkpoint adaptation, a distinct phenomenon which occurs after prolonged checkpoint arrest (22). Further, we focus on the process maintaining arrest in irradiated G₂-phase cells and do not consider how arrest is maintained in irradiated S-phase cells that progress into G₂ phase. (Previous studies have shown that while G₂/M arrest is ATM dependent at early times post-IR, at later times it becomes ATR dependent as S-phase cells progress into G₂ phase [2, 33].) To focus on mechanisms maintaining ATM-dependent signaling in G₂-phase cells, we use aphidicolin (APH) to prevent S-phase cells from progressing into G₂ during analysis. We, thus, examine checkpoint maintenance in cells irradiated in G₂ phase and do not evaluate arrest regulated by ATR following replication fork stalling. The basis for our work stems from two recent advances. First, we evaluate the impact of ATM-mediated ATR activation in the light of recent findings that resection occurs in G₂ phase (11). Second, we consider the finding that NHEJ represents the major DSB repair mechanism in G₂ and that a 15 to 20% subset of DSBs, representing those that are rejoined with slow kinetics in an ATM-dependent manner, undergo resection and repair by HR (3, 25). Thus, contrary to the notion that HR represents the major DSB repair pathway in G₂ phase, it repairs only 15 to 20% of X- or gamma-ray-induced DSBs and represents the slow component of DSB repair in G₂ phase. Given these findings, several potential models for how checkpoint arrest is maintained in G₂ can be envisaged. A simple model is that the initial signal generated by IR is maintained for a defined time to allow for DSB repair. Such a model appears to explain the kinetics of checkpoint signaling in fission yeast after moderate IR (17). In mammalian cells, the duration of arrest depends on dose and DSB repair capacity (6). Thus, it is possible that the status of ongoing repair is communicated to the checkpoint machinery to coordinate timely release with the process of DSB repair. Here, we consider the impact of resection leading to ATM-ATR-Chk1 signaling versus ATM-Chk2 signaling from nonresected DSBs and how they interplay to maintain rather than initiate checkpoint arrest.Mediator proteins, including 53BP1 and MDC1, assemble at DSBs in an ATM-dependent manner, but their roles in the DDR are unclear. Cells lacking 53BP1 or MDC1 are proficient in checkpoint initiation after moderate IR doses, leading to the suggestion that these proteins are required for amplification of the ATM signal after exposure to low doses but are dispensable after high doses, when a robust signal is generated, even in their absence (7, 16, 28, 31). Despite their apparent subtle role in ATM signaling, cells lacking these mediator proteins display significant genomic instability (19). We thus also examine whether the mediator proteins contribute to the maintenance of checkpoint arrest.We identify two ATM-dependent processes that contribute to the maintenance of checkpoint arrest in G₂-phase cells: (i) ATR-Chk1 activation at resected DSBs and (ii) a process that involves sustained signaling from ATM to Chk2 at unrepaired DSBs. Further, we show that 53BP1 and MDC1 are required for maintaining checkpoint arrest, even following exposure to high radiation doses due to roles in ATR-Chk1 activation and sustained ATM-Chk2 signaling, and that this contributes to their elevated genomic instability.     

In vitro establishment of human fibroblasts of lysosomal diseases,GM1-gangliosidosis and Sandhoff disease,by transformation with origin-minus SV40 DNA

The permanent human cell lines preserving defects of lysosomal enzymes, G_M1-1019-SV and SA-1077-SV, were established from the respective fibroblasts from patients with G_Ml-gangliosidosis and Sandhoff disease by transfection with replication origin-minus simian virus 40 DNA. These ceils grow rapidly without entering senescence during more than 120 population doublings. The activity of -galactosidase in G_M1-1019-SV and of B-N-acetylhexosaminidase in SA-1077-SV was respectively 40- and 180-fold lower than that of normal fibroblasts.     

The behavior of allocyclic chromosomes in Bloom's syndrome

The behavior of individual allocyclic chromosomes has been analyzed in lymphocytes of a sister and a brother with Bloom's syndrome. Of 4,633 diploid cells, 115 showed allocyclic chromosomes, and 74 of these had 44, 45 or 46 normal metaphase chromosomes accompanied by one or two allocyclic chromosomes. Of 56 tetraploid cells, 9 contained such chromosomes. The allocyclic chromosomes appeared pulverized or extended corresponding to S or G₂ PCC. We have proposed the hypothesis that individual allocyclic chromosomes do not, as a rule, come from micronuclei, as has often been assumed, but have been left behind in their cycle. This would be caused by a mutation or deletion of a hypothetical coiling center situated near the centromere of each chromosome arm. The following observations agree with our explanation but less well or not at all with the idea of micronuclei: (1) In only 9.6% of the cells does the allocyclic chromosome lie at the edge of the metaphase plate. (2) In 24 cells a part of a chromosome is pulverized while the rest is in metaphase. (3) Both a pulverized and an extended chromosome were present in the same cell. (4) A pulverized acrocentric is often nose-to-nose with a normal D or G chromosome. (5) No allocyclic chromosomes corresponding to G₁ PCC have been found in our material. (6) When a ring is replaced by an allocyclic chromosome, it is usually a member of a 46-chromosome complement. Furthermore, the occurrence of allocyclic chromosomes is correlated with that of other chromosome anomalies which do not follow a Poisson distribution. Allocyclic chromosomes are also more frequent (16%) in tetraploid than in diploid cells (2%).     

Effect of caffeine in Fanconi anemia

Summary In Fanconi anemia (FA) cells the duration of the G₂ phase of the cell cycle prolonged. Such a slowing of the G₂ phase can be induced in normal cells by irradiation with rays during S phase, which also further increases the duration of G₂ in FA cells. The addition of caffeine during the last 7h of culture shortens the G₂ phase in both nonirradiated and irradiated FA cells. In nonirradiated normal cells it may have no effect or may increase G₂ phase duration, but in irradiated normal reduces the slowing of G₂ induced by the radiation. This suggests that FA cells recognize and repair preexisting DNA lesions during G₂ phase and that caffeine inhibits this process. The principal anomaly in FA may be a deficient repair during S phase, as manifest in the prolonged postreplication repair period during G₂ phase required to repair the larger number of lesions passing through S phase.     

The assessment of cellular proliferation by immunohistochemistry: A review of currently available methods and their applications

Summary Immunohistochemical methods using antibodies to cell cycle-related antigens may be used as a means of assessing various aspects of proliferation in tissue, and have the important advantage of preserving the spatial orientation of proliferating cells in histological sections. Currently, the most widely available antibodies for this purpose are antibodies to bromodeoxyuridine (BrdU), Ki67 and antibodies to proliferating cell nuclear antigen (PCNA). BrdU is a thymidine analogue incorporated during the S phase of the cell cycle, which can be introduced by in vivo administration or by in vitro incubation, and monoclonal antibodies are available to display its localization. Ki67 demonstrates a nuclear antigen expressed in all phases of the cell cycle, except G₀ and early G₁, but can only be applied to frozen tissue. PCNA is a nuclear antigen which is essential for DNA synthesis, two commercially available antibodies to PCNA work in paraffin-embedded tissue, but may have different staining characteristics under different conditions of fixation. The main advantages and disadvantages of these different techniques are discussed, together with their main applications to date.     

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

Background

Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells.

Results

In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G₀/G₁ and Sub-G₁ cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G₀/G₁ phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment.

Conclusions

The results demonstrated that HCT-induced G₀/G₁ phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells     

Reduction in Gh protein expression is associated with cytodifferentiation of vascular smooth muscle cells

G_h, a high molecular weight GTP-binding protein that couples ₁-adrenoceptors in heart and liver to phosphatidylinositol (Ptdlns)-specific phospholipase C (PLC), has recently been shown to be a tissue transglutaminase type 11. Transglutaminases have been suggested to play a role in the maintenance of blood vessel structure, and therefore it is possible that changes in their expression may accompany pathological states which involve phenotypic modulation of smooth muscle. Hence, we investigated the expression of G_h during differentiation of rat aortic smooth muscle cells in culture. G_h content was reduced markedly in cultured smooth muscle cells compared to freshly isolated cells as determined by Western blotting using a G_h-specific monoclonal antibody. In contrast, the level of G_q, a heterotrimeric G-protein that couples ₁-adrenoceptors to PLC, was maintained throughout the culture period. These findings indicate that changes in G, expression accompany phenotypic modulation of vascular smooth muscle cells. These changes in G_h protein expression may be important in the altered responsiveness of vessels in pathological disease states.     

Expression of neutral glycosphingolipids and gangliosides in human skeletal and heart muscle determined by indirect immunofluorescence staining

The expression of neutral glycosphingolipids and gangliosides has been studied in human skeletal and heart muscle using indirect immunofluorescence microscopy. Transversal and longitudinal cryosections were immunostained with specific monoclonal and polyclonal antibodies against the neutral glycosphingolipids lactosylceramide, globoside, Forssman glycosphingolipid, gangliotetraosylceramide, lacto-N-neotetraosylceramide and against the gangliosides G_M3(Neu5Ac) and G_M1(Neu5Ac). To confirm the lipid nature of positive staining, control sections were treated with methanol and chloroform:methanol (1:1) before immunostaining. These controls were found to be either negative or strongly reduced in fluorescence intensity, suggesting that lipid bound oligosaccharides were detected. In human skeletal muscle, lactosylceramide was found to be the main neutral glycosphinogolipid. Globoside was moderately expressed, lacto-N-neotetraosylceramide and gangliotetraosylceramide were minimally expressed and Forssman glycosphingolipid was not detected in human skeletal muscle. The intensities of the immunohistological stains of G_M3 and G_M1 correlated to the fact that G_M3 is the major ganglioside in skeletal muscle whereas G_M1 is expressed only weakly. In human heart muscle globoside was the major neutral glycosphingolipid. Lactosylceramide and lacto-N-neotetraosylceramide were moderately expressed, gangliotetraosylceramide was weakly expressed and the Forssman glycosphingolipid was not expressed at all in cardiac muscle. G_M3 and G_M1 were detected with almost identical intensity. All glycosphingolipids were present in plasma membranes as well as at the intracellular level. Abbreviations used: BSA, bovine serum albumin; DAPI, 4,6-diamidine-2-phenylindole-dihydrochloride; DTAF, fluorescein isothiocyanate derivative; GSL(s), glycosphingolipid(s); Neu5Ac,N-acetylneuraminic acid [50]; PBS, phosphate buffered saline. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [51] and the nomenclature of Svennerholm [52]. Lactosylceramide or LacCer, Gal1-4Glc1-1Cer; gangliotriaosylceramide or GgOse₃Cer, GalNAc1-4Gal1-4Glc1-1Cer; globotriaosylceramide or GbOse₃Cer, Gall-4Gall-4Glcl-1Cer; gangliotetraosylceramide or GgOse₄Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; globotetraosylceramide or GbOse₄Cer, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; lacto-N-neotetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; Forssman GSL or GbOse₃Cer, GalNAc1-3GalNAc1-3Gal1-4Gal1-4Gle1-1Cer; G_M3, II³Neu5Ac-LacCer; G_M2, II³Neu5Ac-GgOse₃Cer; G_M1, II³Neu5Ac-GgOse₄Cer; G_D3 II³(Neu5Ac)₂-LacCer; G_D2, II³(Neu5Ac)₂-GgOse₃Cer; G_D1a, IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer; G_D1b, II³(Neu5Ac)₂-GgOse₄Cer.