Combination Of Actinomycin Research Articles

The Strong Activation of p53 Tumor Suppressor Drives the Synthesis of the Enigmatic Isoform of DUSP13 Protein.

The p53 tumor suppressor protein activates various sets of genes depending on its covalent modifications, which are controlled by the nature and intensity of cellular stress. We observed that actinomycin D and nutlin-3a (A + N) collaborate in inducing activating phosphorylation of p53. Our recent transcriptomic data demonstrated that these substances strongly synergize in the upregulation of DUSP13, a gene with an unusual pattern of expression, coding for obscure phosphatase having two isoforms, one expressed in the testes and the other in skeletal muscles. In cancer cells exposed to A + N, DUSP13 is expressed from an alternative promoter in the intron, resulting in the expression of an isoform named TMDP-L1. Luciferase reporter tests demonstrated that this promoter is activated by both endogenous and ectopically expressed p53. We demonstrated for the first time that mRNA expressed from this promoter actually produces the protein, which can be detected with Western blotting, in all examined cancer cell lines with wild-type p53 exposed to A + N. In some cell lines, it is also induced by clinically relevant camptothecin, by nutlin-3a acting alone, or by a combination of actinomycin D and other antagonists of p53-MDM2 interaction-idasanutlin or RG7112. This isoform, fused with green fluorescent protein, localizes in the perinuclear region of cells.

Transcriptomic and proteomic study of cancer cell lines exposed to actinomycin D and nutlin-3a reveals numerous, novel candidates for p53-regulated genes

Transcriptomic analyses have revealed hundreds of p53-regulated genes; however, these studies used a limited number of cell lines and p53-activating agents. Therefore, we searched for candidate p53-target genes by employing stress factors and cell lines never before used in a high-throughput search for p53-regulated genes. We performed RNA-Seq on A549 cells exposed to camptothecin, actinomycin D, nutlin-3a, as well as a combination of actinomycin D and nutlin-3a (A + N). The latter two substances synergise upon the activation of selected p53-target genes. A similar analysis was performed on other cell lines (U-2 OS, NCI–H460, A375) exposed to A + N. To identify proteins in cell lysates or those secreted into a medium of A549 cells in control conditions or treated with A + N, we employed mass spectrometry. The expression of selected genes strongly upregulated by A + N or camptothecin was examined by RT-PCR in p53-deficient cells and their controls. We found that p53 participates in the upregulation of: ACP5, APOL3, CDH3, CIBAR2, CRABP2, CTHRC1, CTSH, FAM13C, FBXO2, FRMD8, FRZB, GAST, ICOSLG, KANK3, KCNK6, KLRG2, MAFB, MR1, NDRG4, PTAFR, RETSAT, TMEM52, TNFRSF14, TRANK1, TYSND1, WFDC2, WFDC5, WNT4 genes. Twelve of these proteins were detected in the secretome and/or proteome of treated cells. Our data generated new hypotheses concerning the functioning of p53. Many genes activated by A + N or camptothecin are also activated by interferons, indicating a noticeable overlap between transcriptional programs of p53 and these antiviral cytokines. Moreover, several identified genes code for antagonists of WNT/β-catenin signalling pathways, which suggests new connections between these two cancer-related signalling systems. One of these antagonists is DRAXIN. Previously, we found that its gene is activated by p53. In this study, using mass spectrometry and Western blotting, we detected expression of DRAXIN in a medium of A549 cells exposed to A + N. Thus, this protein functions not only in the development of the nervous system, but it may also have a new cancer-related function.

Staggered intercalation of DNA duplexes with base-pair modulation by two distinct drug molecules induces asymmetric backbone twisting and structure polymorphism

The use of multiple drugs simultaneously targeting DNA is a promising strategy in cancer therapy for potentially overcoming single drug resistance. In support of this concept, we report that a combination of actinomycin D (ActD) and echinomycin (Echi), can interact in novel ways with native and mismatched DNA sequences, distinct from the structural effects produced by either drug alone. Changes in the former with GpC and CpG steps separated by a A:G or G:A mismatch or in a native DNA with canonical G:C and C:G base pairs, result in significant asymmetric backbone twists through staggered intercalation and base pair modulations. A wobble or Watson–Crick base pair at the two drug-binding interfaces can result in a single-stranded ‘chair-shaped’ DNA duplex with a straight helical axis. However, a novel sugar-edged hydrogen bonding geometry in the G:A mismatch leads to a ‘curved-shaped’ duplex. Two non-canonical G:C Hoogsteen base pairings produce a sharply kinked duplex in different forms and a four-way junction-like superstructure, respectively. Therefore, single base pair modulations on the two drug-binding interfaces could significantly affect global DNA structure. These structures thus provide a rationale for atypical DNA recognition via multiple DNA intercalators and a structural basis for the drugs’ potential synergetic use.

M23 - GENETIC ASSOCIATION AND FUNCTIONAL CHARACTERIZATION OF A VARIANT IN THE MCPH1 GENE IN BIPOLAR DISORDER AND SCHIZOPHRENIA

Background Both Schizophrenia (SCZ) and Bipolar Disorder (BPD) disorder are serious psychiatric conditions that can give rise to debilitating symptoms that can severely impact the life of the affected individual, and their families. We have previously reported that the MCPH1 gene variant rs61749465 A>G (p.Asp61Gly) showed suggestive evidence for association with schizophrenia (SCZ; Leonenko et al. 2017). The MCPH1 gene has multiple roles in cellular functions including, DNA damage/repair pathways; centrosomal localization; E2F transcription factor 1-mediated apoptosis; transcriptional activation of cell cycle checkpoint, DNA stability; and telomere structure. A second MCPH1 point mutation (rs199422124 C>G; p.Thr27Arg) is an autosomal recessive cause of microcephaly. Methods The schizophrenia associated MCPH1 variant rs61749465 was tested for association in 2,300 Bipolar Disorder (BPD) subjects, and 1,820 normal comparison subjects. We also analysed the microcephaly causing mutation rs199422124 in the BPD and control subjects and in our SCZ cohort of 1,930 subjects. Next, we conducted analysis of the in vitro effects of the rs6174965 and rs199422124 variants on cell survival/proliferation using cell counts, and the MTT assay; their effect on DNA damage using the comet assay; their effect on mRNA stability using a combination of Actinomycin D treatment followed by Q-PCR; and their effect on gene expression using RNA-Seq analysis combined with pathway analysis and gene network analyses. Results After genotyping we report evidence for association with BPD (P=0.0009). Notably the variant allele of rs61749465 was absent in the 1,820 comparison subjects tested. rs61749465 is located in the N-terminal of the BRCT1 domain of MCPH1. Bioinformatic analysis predicted the Asp61Gly substitution to be damaging to MCPH1 protein function. rs199422124 was not detected in any of the participants. We sought to characterize the functional effects of these variants on MCPH1 function. Cell viability and cell count assays indicated that the variant allele of rs199422124 had a larger impact on cell survival compared to the variant allele of rs61749465, however neither of the variant alleles significantly altered DNA damage or mRNA stability. Gene expression analysis for rs61749465 using RNA-seq shows that the expression of a number of heat shock protein have been affected. Gene network and pathway analysis indicated that the variant alleles may impact cellular aging and protein translation. Discussion Further in vitro and in vivo characterization of the rs61749465 variant in MCPH1 may provide insight into the molecular pathogenesis of psychosis.

Cytoprotective effect of γ-tocopherol against tumor necrosis factor α induced cell dysfunction in L929 cells

The antioxidant vitamin γ-tocopherol exerts protective and anti-inflammatory effects in various models of critical illness. The combination of actinomycin D and tumor necrosis factor α (TNFα) in the immortalized fibroblast cell line L929 is a well-established method to model pro-inflammatory cytotoxicity in cultured cells in vitro. The present study had two aims. First, we wished to characterize the contribution of reactive oxygen species (ROS) to the cell dysfunction and this commonly used model system of cell death. Second, we wished to investigate the effects of γ-tocopherol on this response. Cells were exposed to actinomycin D (0.5 µg/ml) + TNFα (100 pg/ml) in the absence or presence of 1 h of γ-tocopherol pre-treatment. The earliest change that was detected in our system in response to TNFα was an increase in mitochondrial oxidant production, already apparent at 45 min. Changes in glycolysis and oxidative phosphorylation parameters were already apparent at 2 h, as detected by the Seahorse Biosciences XF24 Flux Analyzer. By 6 h, a slight decrease in Cell Index was detected by impedance-based analysis, employing an electronic sensor array system (XCelligence). At the same time, a slight decrease in cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method, along with a significant increase in lactate dehydrogenase (LDH) release into the culture medium, and a detectable degree of mitochondrial membrane depolarization. Between 12 and 24 h, the cell viability (already at a low level) further declined, which coincided with a secondary, marked decline in the mitochondrial membrane potential. Pre-treatment of the cells with γ-tocopherol (10-300 µM) provided a significant protection against all of the functional alterations induced by actinomycin D and TNFα. The current study provides direct evidence that reactive oxidant formation plays an important role in the current experimental model of cell dysfunction, and demonstrates the protective effects of the potent endogenous antioxidant vitamin, γ-tocopherol. The mechanisms described in the current study may, in part, contribute to the protective effects of γ-tocopherol in various models of critical illness.

Actinomycin D synergistically enhances the efficacy of the BH3 mimetic ABT-737 by downregulating Mcl-1 expression

Many types of cancer cells possess the ability to evade apoptosis, leading to their rapid and uncontrolled proliferation. As major regulators of apoptosis, Bcl-2 proteins serve as emerging targets for novel chemotherapeutic strategies. In this study, we examined the involvement of Bcl-2 proteins in apoptosis induced by the chemotherapeutic agent actinomycin D. A dramatic decrease in anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) mRNA and protein expression was detected upon actinomycin D treatment. Further, Mcl-l over-expression caused resistance to cell death upon treatment with actinomycin D, implicating a role for the down-regulation of Mcl-1 in actinomycin D-induced apoptosis. We also explored the therapeutic potential of actinomycin D in combination with ABT-737, an experimental agent that inhibits anti-apoptotic Bcl-2 proteins. Actinomycin D sensitized cells to ABT-737 treatment in a Bak- or Bax-dependent manner. Importantly, low concentrations of actinomycin D and ABT-737 were more effective in inducing cell death in transformed cells than their untransformed counterparts. A synergistic effect of actinomycin D and ABT-737 on cell death was observed in several human tumor cell lines. Like actinomycin D treatment, knocking down Mcl-1 expression greatly sensitized tumor cells to ABT-737, and Mcl-1 over-expression abrogated the cytotoxic effect induced by ABT-737 and actinomycin D. These results suggest that the down-regulation of Mcl-1 by actinomycin D is likely responsible for the observed synergistic effect between the two drugs. Overall, our studies provide compelling evidence that the combination of actinomycin D and ABT-737 may lead to an effective cancer treatment strategy.

Feasibility of chemosensitivity testing in soft tissue sarcomas

BackgroundSoft tissue sarcomas comprise less than 1% of all solid malignancies. The presentation and behavior of these tumors differs depending on location and histological characteristics. Standard therapy consists of complete surgical resection in combination with adjuvant radiotherapy. The role of chemotherapy is not clearly defined and is largely restricted to clinical trials. Only a limited number of agents have proved to be effective in soft tissue sarcomas. The use of doxorubicin, epirubicin and ifosfamide allowed response rates of more than 20%. In addition, recent chemotherapy trials did not demonstrate any significant differences in efficacy for various histological subtypes.MethodsThe objective of this study was to gain additional information about the chemosensitivity of soft tissue sarcomas to seven 7 different chemotherapy agents as single drugs and 4 combinations. Therefore we used an established ATP based in-vitro testing system and examined 50 soft tissue sarcomas. Chemosensitivity was assessed using a luciferin-luciferase-based luminescence assay providing individual chemosensitivity indices for each agent tested.ResultsThe sensitivity varied widely according to the histological subtypes. The tumors state of cellular dedifferentiation played a crucial role for the efficiency of the chemotherapeutic agents. The sensitivity also depended on the presentation of the sarcoma as a primary or recurrent tumor. The highest sensitivity was demonstrated for actinomycin D as a single agent, with 74% of the tumor samples exhibiting a high-grade sensitivity (20% low sensitivity, no resistance). The combination of actinomycin D and ifosfamide yielded a high sensitivity in 76% (2% resistance). Doxorubicin as a mono-therapy or in combination with ifosfamide achieved high sensitivity in 70% and 72%, respectively, and resistance in 6% of the samples.ConclusionChemosensitivity testing is feasible in soft tissue sarcomas. It can be used to create sensitivity and resistance profiles of established and new cytotoxic agents and their combinations in soft tissue sarcomas. Our data demonstrate measurable discrepancies of the drug efficiency in soft tissue sarcomas, sarcoma subtypes and tumor recurrencies. However, current therapeutic regime does not take this in consideration, yet.

Augmentation of antitumor efficacy by the combination of actinomycin D with tumor necrosis factor-alpha and interferon-gamma on a melanoma model in mice.

The efficacy of combination treatment with actinomycin D (Act D), recombinant human tumor necrosis factor-alpha (TNF-alpha), and recombinant murine interferon-gamma (IFN-gamma) was examined on established MmB16 melanoma in mice. TNF-alpha alone had marginal effect in vitro on melanoma cells. However, when this cytokine was combined with either Act D or IFN-gamma, synergistic cytostatic/cytotoxic effects were observed. The highest cytotoxicity was demonstrated in cultures of melanoma cells in which all three agents together were added. In mice inoculated with 10(6) melanoma cells (into the footpad of the hind limb) and treated locally with Act D, TNF-alpha and IFN-gamma, beneficial therapeutic effects were found. When initiated 1 week after tumor cell inoculation, the 7-day treatment with all these agents administered together at daily doses: 0.2 microgram (Act D), 1 microgram (TNF-alpha), and 200 U (IFN-gamma) resulted in a significant delay of tumor progression in comparison to the therapy that included either Act D alone or TNF-alpha in combination with IFN-gamma. Side effects of such a treatment, both local and systemic, were negligible. The results of this study demonstrate that combination of regional chemotherapy (actinomycin D) and immunotherapy (TNF-alpha/IFN-gamma) may display higher efficacy than either treatment alone and may increase therapeutic index without augmenting toxic effects.

ADP-Ribosylation Inhibitors Inhibit Cellular RNA Synthesis but Do Not Affect Expression of Manganous Superoxide Dismutase or Heat Shock Protein 70 in Tumor Necrosis Factor αSensitive and -Resistant Tumor Cells

We have shown that the cytotoxic response of TNF-sensitive L929 cells and TNF-resistant EMT-6 cells to TNF-alpha can be modulated by ADP-ribosylation inhibitors independently of ADP-ribosylation rates. To explore the possibility that these inhibitors modulate TNF cytotoxicity by interfering with cellular protective mechanisms, we evaluated their effects on general RNA synthesis and on mRNA expression of two proposed protective genes, manganous superoxide dismutase (MnSOD) and heat shock protein 70 (hsp70). We found that ADP-ribosylation inhibitors could inhibit general RNA synthesis in a dose-dependent fashion to a similar extent in both EMT-6 and L929 cells, although these inhibitors increased or decreased the sensitivity of the cells to TNF, respectively. In EMT-6 cells, combination of actinomycin D with these inhibitors further inhibited the RNA synthesis rate, and it actually decreased the TNF sensitivity of the EMT-6 cells. Furthermore, the expression of MnSOD or hsp70 was not regulated by these inhibitors. Thus, TNF resistance must depend on other mechanisms in addition to the expression of these protective genes.

Phase II study of a combination of actinomycin (A) and etoposide (E) in good risk (GR) trophoblastic disease (TD)

Phase II study of a combination of actinomycin (A) and etoposide (E) in good risk (GR) trophoblastic disease (TD)

Phase II evaluation of dibromodulcitol and actinomycin D, hydroxyurea, and cyclophosphamide in previously untreated patients with malignant melanoma.

In this Eastern Cooperative Oncology Group (ECOG) phase II study, dibromodulcitol (DBD) and a combination of actinomycin D, hydroxyurea, and cyclophosphamide (AHC) were compared with methyl-CCNU, the current ECOG standard, in patients who had received no prior chemotherapy for disseminated malignant melanoma. The response rates were 6% (3/50) for AHC, 9% (3/34) for DBD, and 14% (7/49) for methyl-CCNU. Median survival times were 4, 5, and 6 months, respectively. Neither regimen appears to offer any advantage over methyl-CCNU as front-line therapy for patients with disseminated melanoma.

Treatment of advanced colorectal carcinoma with actinomycin D, vincristine, methyl-CCNU, and methotrexate.

Twenty patients with advanced colorectal carcinoma were treated with combination chemotherapy consisting of actinomycin D, vincristine, methyl-CCNU, and methotrexate. Fourteen patients had received prior chemotherapy with 5-fluorouracil (5-FU). No complete responses and only one partial response were observed for an overall response rate of 5%. The combination of actinomycin D, vincristine, methyl-CCNU, and methotrexate at the doses and schedule used was of no value in the treatment of patients with metastatic colorectal carcinoma.

Messenger RNA half-life in Dictyostelium discoideum

Messenger RNA half-life in vegetatively growing cells of Dictyostelium discoideum was determined using a uridine pulse-chase procedure. In these experiments, mRNA decayed in a complex fashion, and consisted of at least two major components, one with a half-life of about 50 min and a second with a half-life of about 10 hr. These results independently confirm our previous studies on the decay of mRNA extracted from actinomycin D-treated cells. Since these results were in apparent conflict with half-life determinations obtained with a combination of actinomycin D and daunomycin ( J. P. Margolskee and H. F. Lodish, 1980a, Dev. Biol. 74, 37–49), we have also studied mRNA half-life in cells treated with a combination of both drugs and found that simultaneous use of both drugs leads to accelerated mRNA decay and other noticeable side effects. In light of our observations. we have suggested an alternative to conclusions drawn by others with respect to mRNA synthesis and stability in Dictyostelium development.

Regulation of ornithine decarboxylase during morphogenesis of Mucor racemosus.

During the yeast-to-hyphae transition of the dimorphic phycomycete Mucor racemosus, there was a 30- to 50-fold increase in the activity of ornithine decarboxylase. Increased enzyme activity preceded the emergence of germ tubes and reached a maximum before conversion was completed. Subsequently, enzyme levels rapidly declined, despite the continuation of mycelial growth. Both putrescine and spermidine blocked the enzyme activity response. Protein synthesis was required for the increase in enzyme activity during morphogenesis. A combination of actinomycin D and netropsin inhibited ribonucleic acid synthesis but failed to inhibit the increase in ornithine decarboxylase activity. There was a twofold increase in the enzyme half-life during morphogenesis with either trichodermin or verrucarin to inhibit protein synthesis.

Evaluation of cytotoxic agents in human colonic tumor xenografts and mouse gastrointestinal tissues using a 3H-thymidine fractional incorporation assay

Drug induced changes in 3H -thymidine fractional incorporation (TFI) in 4 human colonic xenograft lines and mouse gastrointestinal tissues have been related to the growth inhibition and host toxicity respectively. Using the TFI assay, the combinations of actinomycin D with cyclophosphamide and methyl CCNU with pentamethyl-melamine have been examined. Both combinations showed greater than additive activity against only 1 of the 3 xenograft lines in which they were tested. The TFI assay allows more rapid evaluation of drug activity and is more sensitive than growth delay studies. It also allows quantitative measurement of cytotoxic activity in gastrointestinal tissues after sublethal dose levels.

Origin of the actinomycin D insensitive RNA species in Aedes albopictus cells.

In the presence of actinomycin D or a combination of actinomycin D and either camptothecin or alpha-amanatin. Aedes albopictus cells synthesize a variety of single stranded RNA species. These actinomycin D resistant species are ethidium bromide sensitive and they are present in the cell cytoplasm in an RNase resistant structure which has the sedimentation and buoyant density characteristics of mitochondria. Twelve actinomycin D insensitive RNA species can be detected by electrophoresis in 7M urea and 11 of these bind to oligo(dT)-cellulose. An identical set of oligo(dT)-cellulose binding RNA species is obtained when A. albopictus cells are labeled in the presence of camptothecin alone. The actinomycin D insensitive RNA species which bind to oligo(dT)-cellulose hybridize to mitochondrial DNA. These data indicate that the actinomycin D insensitive RNA species have a mitochondrial origin and are not associated with the replication of an inapparent contaminating virus.

Synthesis of single- and double-stranded cucumber mosaic virus RNAs in tobacco mesophyll protoplasts

Five major RNAs were isolated by polyacrylamide gel electrophoresis from purified virions of the yellow strain of cucumber mosaic virus (CMV-Y), although the smallest RNA (RNA 5) was not detectable in RNAs of the ordinary strain of CMV (CMV-O). Synthesis of CMV-specific RNAs was investigated using tobacco mesophyll protoplasts inoculated with CMV in vitro . Incorporation of [ 3 H]uracil into RNAs was analyzed by electrophoresis in polyacrylamide gels. A combination of actinomycin D treatment and ultraviolet irradiation to protoplasts showed a marked inhibiting effect on host RNA synthesis, but little on viral RNA synthesis. Replication of the five major RNAs of CMV-Y occurred in the protoplasts, although RNAs 1 and 2 could not be resolved; RNAs 3 and 5 were especially predominant. In the case of CMV-O, RNA 5 was not synthesized in vivo . The infectivity of RNA extracted from CMV-infected tobacco protoplasts was detectable within 6 hr after inoculation, and reached its maximum at 18 hr; it then decreased rather sharply. The rate of viral RNA synthesis was highest at 12 to 18 hr after inoculation, and after that it gradually slowed. Double-stranded RNAs were also isolated from the infected protoplasts. Existence of five pairs of replicative form (RF) and replicative intermediate (RI) RNAs corresponding to the respective single-stranded CMV RNAs was assumed by examining their chromatographic behavior on a cellulose column, solubility in 1 M NaCl and susceptibility to ribonuclease treatment. The smallest RI, 5, was eluted from the cellulose column together with single-stranded RNAs with a buffer containing 15% ethanol. Much larger amounts of RF 3 and 5 were detected as compared with the other RF species, and the electrophoretic pattern of the RFs was very similar to that of single-stranded CMV RNAs syntheiszed in vivo except for the slower electrophoretic mobilities of the RFs.

A Murine Wilms' Tumor as a Model for Chemotherapy and Radiotherapy 2 3

We studied the effect on tumor volumes and host survival of twenty-four chemotherapeutic and radiotherapeutic regimens in rats bearing the Furth/Columbia murine Wilms' tumor. After 5 years and some 100 transfers, this tumor retained its sensitivity to suitable combinations of actinomycin D, vincristine, and X-ray, regimens in current use in the treatment of human Wilms' tumor. Cytoxan alone was highly inhibitory; adriamycin was modestly inhibitory; and lethal doses of methotrexate followed by leucovorin "rescue" were slightly inhibitory to the growth of the rat tumor. No other chemotherapeutic agent tested was effective by our criteria. Cytoxan, with or without adriamycin, and X-irradiation, were the most useful combinations studied. The chemotherapeutic agents given 3 days before irradiation were significantly more carcinostatic than they were when given after radiotherapy.

Radiation and actinomycin D mortality studies in C57BL 6 mice. II. Combined treatment and gastrointestinal lethality.

C57BL/6 mice were treated with actinomycin or radiation therapy and with a combination of the two agents. LD50 estimates based on radiation doses were calculated from 6-day (intestinal death) mortality data. A factorial analysis of the data was performed to determine the. main effects for actinomycin (A) or radiation exposure (R) as well as actinomycin-radiation (AR) interactions. The LD50 data indicated that the effects of the combined treatment were generally more lethal in causing intestinal death than the effects of either agent given independently. Significant main effects for A and R were generally observed with few AR interactions. The principal mechanism for intestinal death in animals treated with various combinations of actinomycin and radiation exposure appears to be additive cytotoxicity either at the level of crypt survival and/or prolongation of the time to the appearance of the compensatory proliferative response.

Actinomycin D and the regulation of enzyme biosynthesis during development of Dictyostelium discoideum

Actinomycin D, when added to differentiating Dictyostelium discoideum cells, completely inhibits synthesis of ribosomal RNA but only partially inhibits synthesis of mRNA. Hence, earlier experiments using this drug, which indicated that the period of synthesis of mRNAs for several developmentally regulated enzymes preceded the period of translation by several hours, must be re-evaluated. We show here that a combination of actinomycin D and daunomycin does rapidly inhibit synthesis of mRNA; also there is a rapid and complete inhibition of biosynthesis of the three developmentally regulated enzymes studied. One interpretation of these results is that translation of mRNA follows closely upon synthesis of the mRNA, and that the primary control of protein biosynthesis during Dictyostelium development is at the level of mRNA transcription.