Identification and characterization of a novel Mdm2 splice variant acutely induced by the chemotherapeutic agents Adriamycin and Actinomycin D

Mdm2, as the most important negative regulator of p53, plays an important homeostatic role in regulating cell division and the cellular response to DNA damage, oncogenic insult, and other forms of cellular stress. We discovered that the DNA damaging agent adriamycin (doxorubicin) induces a novel aberrantly spliced Mdm2 mRNA which incorporates 108bp of intronic sequence not normally found in the Mdm2 mature mRNA. Accordingly, we term this Mdm2 splice variant Mdm2+108. Importantly, this insertion introduces in-frame nonsense codons, thus encoding a profoundly truncated mdm2 protein lacking the C-terminal RING finger domain and the E3 ubiquitin ligase activity. A wide range of pharmacological testing revealed that Mdm2+108 is induced, in mouse and rat cells, in specific response to Adriamycin and actinomycin D, but not other modes of DNA damage. Meanwhile, antibodies against the N-terminal region of mdm2 reveal a marked reduction in detectable mdm2 protein upon Adriamycin treatment, while p53 accumulates to strikingly high levels. We thus conclude that this alternative spicing of Mdm2 may be an important mechanism to facilitate massive accumulation of p53 in response to genotoxic agents.     

Malignant H1299 tumour cells preferentially internalize iron-bound inositol hexakisphosphate

In colon enterocytes and in well-differentiated colon cancer CaCo-2 cells, InsP₆ (inositol hexakisphosphate) inhibits iron uptake by forming extracellular insoluble iron/InsP₆ complexes. In this study, we confirmed that CaCo-2 cells are not able to take up iron/InsP₆ but, interestingly, found that the cells are able to internalize metal-free and Cr³⁺-bound InsP₆. Thus, the inability of CaCo-2 cells to take up iron/InsP₆ complexes seems to be due to the iron-bound state of InsP_6. Since recently we demonstrated that the highly malignant bronchial carcinoma H1299 cells internalize and process InsP_6, we examined whether these cells may be able to take up iron/InsP₆ complexes. Indeed, we found that InsP₆ dose-dependently increased uptake of iron and demonstrated that in the iron-bound state InsP₆ is more effectively internalized than in the metal-free or Cr³⁺-bound state, indicating that H1299 cells preferentially take up iron/InsP₆ complexes. Electron microscope and cell fraction assays indicate that after uptake H1299 cells mainly stored InsP₆/iron in lysosomes as large aggregates, of which about 10% have been released to the cytosol. However, this InsP₆-mediated iron transport had no significant effects on cell viability. This result together with our finding that the well-differentiated CaCo-2 cells did not, but the malignant H1299 cells preferentially took up iron/InsP_6, may offer the possibility to selectively transport cytotoxic substances into tumour cells.     

Caffeine induces cytoskeletal changes and cell death in H1299 cells

Caffeine is the most common natural neuroactive substance around the world. The exact mechanism of the anticancer effects of caffeine is not clear, especially in the contexts of the cytoskeletal changes. It is known that caffeine exerts an effect on cell cycle, cell proliferation, radiosensivity of cells, and also induces cell death. The aim of the study was to determine the effect of 10 and 20 mM L^?1 caffeine on the major cytoskeletal proteins in non-small lung cancer cell line H1299. Caffeine treatment induced abnormalities in morphology and ultrastructure of cells. Moreover, the fluorescence studies showed changes in organization of vimentin, β-tubulin, lamin A/C and F-actin, which were attributed to the induction of cell death. The results also demonstrated that caffeine induced formation of two cell populations: giant, mono- or multinucleated cells, with the phenotype of mitotic catastrophe and shrunken cells with condensation of chromatin, typical of apoptosis. This study for the first time shows the effect of caffeine on the cytoskeleton of H1299 cell line. In conclusion, a high-dose caffeine treatment induces apoptotic cell death and makes it a powerful anticancer agent that should be considered for the treatment of non-small cell lung cancer.     

Enhancement of radiosensitivity in H1299 cancer cells by actin-associated protein cofilin

Cofilin is an actin-associated protein that belongs to the actin depolymerization factor/cofilin family and is important for regulation of actin dynamics. Cofilin can import actin monomers into the nucleus under certain stress conditions, however the biological effects of nuclear transport are unclear. In this study, we found that over-expression of cofilin led to increased radiation sensitivity in human non-small lung cancer H1299 cells. Cell survival as determined by colony forming assay showed that cells over-expressing cofilin were more sensitive to ionizing radiation (IR) than normal cells. To determine whether the DNA repair capacity was altered in cofilin over-expressing cells, comet assays were performed on irradiated cells. Repair of DNA damage caused by ionizing radiation was detected in cofilin over-expressing cells after 24 h of recovery. Consistent with this observation, the key components for repair of DNA double-strand breaks, including Rad51, Rad52, and Ku70/Ku80, were down-regulated in cofilin over-expressing cells after IR exposure. These findings suggest that cofilin can influence radiosensitivity by altering DNA repair capacity.     

Regulation of p63 function by Mdm2 and MdmX.

p63, a p53-related protein, has been shown to activate p53-responsive genes and induce apoptosis in certain cell types. In this study, we examined the effects of Mdm2 and MdmX proteins on p63 transactivation, apoptosis, and protein levels. The isoforms of p63 most structurally similar to p53, p63gamma (p51A) and p63alpha (p51B), were chosen for study. Our results confirm earlier reports demonstrating that although both p63 isoforms can transactivate p53-responsive promoters and induce apoptosis, p63gamma has a stronger transactivation potential and is a more potent inducer of apoptosis than is p63alpha. In addition, both Mdm2 and MdmX were able to inhibit the transactivation induced by p63gamma and p63alpha. However, only Mdm2 overexpression led to a detectable decrease in p63-induced apoptosis. Although Mdm2 binding to p53 triggers ubiquitin-mediated proteosome degradation, p63 protein levels were unaltered by association with either Mdm2 or MdmX. Finally, immunofluorescence experiments showed that both p63 isoforms were localized in the nucleus and could be exported when coexpressed with Mdm2 but not with MdmX. These findings suggest that both Mdm2 and MdmX can downregulate p63 transactivation potential; however, only Mdm2 is capable of inhibiting the apoptotic function of p63 by removing it from the nucleus.     

Regulation of p53 by Mdm2: fate is in the numbers

The p53 tumor suppressor protein is normally restrained by the Mdm2 oncoprotein, which promotes p53 ubiquitination. In a recent issue of Science, report that p53 may face two alternative fates, depending on Mdm2 levels: high Mdm2 drives p53 polyubiquitination and degradation within the cell nucleus, whereas low Mdm2 promotes p53 monoubiquitination and nuclear exclusion.     

Production of Actinomycin D in Complex Media

The production of actinomycin D was examined using Streptomyces antibioticus growing on a complex medium based on glucose and casein hydrolyzate. Purification of the medium to remove calcium increased actinomycin production by about one-third and prevented production of a characteristic brown pigment. The sensitivity to magnesium concentrations was less than was reported by Katz et al.,¹¹⁾ while similar requirements were found for zinc and iron.     

Ring shaped nucleoli in liver cells of rats after treatment with Actinomycin D

Summary The incidence of ring shaped nucleoli was studied by means of light and electron microscopical methods in liver cells of rats injected intravenously with Actinomycin D in a dose of 150 g per kg body weight.Ring shaped nucleoli were most frequent 20 minutes after administration of Actinomycin D. The number of fragmented nucleoli was increased 20 minutes after administration of the drug and did not decrease even after 72 hours.Fibrillar ring like inclusions within nuclei and more perichromatin granules at the periphery of nucleoli were observed after treatment with Actinomycin D.

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Zusammenfassung Die Wirkung von Aktinomycin D (150 g/kg) auf die Leberzellen der Ratte wurde licht- und elektronenenmikroskopisch untersucht. 20 min nach intravenöser Aktinomycin-D-Gabe wurden ringörmige Nukleolen beobachtet. Die Zahl der fragmentierten Nukleolen war 20 min nach der Applikation gesteigert. Ihre relative Zahl änderte sich nach Aktinomycingabe nicht. Nach Behandlung der Tiere mit Aktinomycin D kann man in den Kernen der Leberzellen fibrilläre ringförmige Einschlüsse und eine größere Menge von perichromatin-granules an der Peripherie der Nukleolen beobachten.

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The authors are indebted to M. Mikoláová, V. Povolná, I. erníková and Dr. M. Titlbach for their help, to Dr. J. Chaloupka for providing Actinomycin D and to Professor Busch and Professor Wolf for many helpful suggestions.     

Actinomycin D induction of acid phosphatase in synchronized 15178Y mouse leukemia cells

Actinomycin D was found to induce synthesis of acid phosphatase activity in L5178Y mouse leukemic cells. The degree of induction was dose-dependent. Furthermore, other RNA synthesis inhibitors (Lucanthone and Daunomycin) were also found to cause an increase in enzyme activity. Such induction of enzyme activity was also found to be dependent on cell cycle position. For up to 5 h after release from Colcemid synchronized cells treated with actinomycin D showed little or no variation from controls. Only when cells were in late S (5th to 8th hours of the cell cycle) could enzyme activity be induced. At this time full induction occurred within 1 h.     

Actinomycin D induced chromosome breakage and suppression of meiosis in the locust,Schistocerca gregaria

Treatment with Actinomycin D has been found to induce chromosome breakage in the spermatocytes of the locust. The broken ends appear not to have the capacity to reunite or to restitute. The treatment with this chemical has also been found to affect the development of the potential spermatocytes so that they fail to enter meiosis. The expected meiotic sequence is found to be replaced by a mitotic division. It has been concluded that both chromosomal aberrations and the effect on development of spermatocytes result from disorganization of the synthetic apparatus of the cells.     

Actinomycin D decreases protein kinase C content and induces neuritogenesis in neuroblastoma cells

We have previously reported that inhibition of protein kinase C induces differentiation of neuroblastoma cells in culture. It is shown now that actinomycin D, a well known inhibitor of DNA synthesis, reduces selectively the content of protein kinase C and induces neuritogenesis in Neuro 2a cells in culture.     

Actinomycin D peritonitis in the rat.

Following intraperitoneal injection of actinomycin D rats show a decrease in number of cells present in the peritoneal cavity, reaching the lowest point after 24 hr. At the same time a highly significant increase of free beta-glucoronidase and of the intracellular concentrations of both cyclic AMP and cyclic GMP has been observed. No exudate was present at this time. Measurable quantities of exudate were present 48-72 hr after actinomycin injection concomitantly with an intense cellular immigration, the dominant cell being mononuclears. In this second phase of the reaction the free beta-glucuronidase decreases towards normal values and both the cyclic nucleotides are significantly below the control values. It is suggested that the increase of intracellular cAMP--concomitant with the maximum release of lysosomal enzymes--is a feedback mechanism preventing further release of inflammatory mediators.     

c-Abl phosphorylation of Mdm2 facilitates Mdm2-Mdmx complex formation

Mdm2 and Mdmx are oncoproteins that have essential yet nonredundant roles in development and function as part of a multicomponent ubiquitinating complex that targets p53 for proteasomal degradation. However, in response to DNA damage, Mdm2 and Mdmx are phosphorylated and protect p53 through various mechanisms. It has been predicted that Mdm2-Mdmx complex formation modulates Mdm2 ligase activity, yet the mechanism that promotes formation of Mdm2-Mdmx complexes is unknown. Here, we show that optimal Mdm2-Mdmx complex formation requires c-Abl phosphorylation of Mdm2 both in vitro and in vivo. In addition, Abl phosphorylation of Mdm2 is required for efficient ubiquitination of Mdmx in vitro, and eliminating c-Abl signaling, using c-Abl(-/-) knock-out murine embryonic fibroblasts, led to a decrease in Mdmx ubiquitination. Further, p53 levels are not induced as efficiently in c-Abl(-/-) murine embryonic fibroblasts following DNA damage. Overall, these results define a direct link between genotoxic stress-activated c-Abl kinase signaling and Mdm2-Mdmx complex formation. Our results add an important regulatory mechanism for the activation of p53 in response to DNA damage.     

Actinomycin D induced DNase I hypersensitivity and asymmetric structure transmission in a DNA hexadecamer.

DNase I cleavage rates and nmr chemical shifts are shown to change for DNA sequences distal to an intercalated actinomycin D molecule in a duplex hexadecamer upon drug binding. Both sets of observations suggest that the source of these changes is a DNA-mediated structural response. The nmr results imply the response is transmitted preferentially in a 5'-to-3' direction from the drug binding site. An inequivalent response of the two strands to a ligand-induced conformational change immediately suggests a mechanism for distinguishing the sense and antisense strands of DNA.