Leon A. G. M. van den Broek

Characterization of the hamster desmin gene: Expression and formation of desmin filaments in nonmuscle cells after gene transfer

The structural organization of the hamster gene encoding the intermediate filament (IF) protein desmin has been determined. The gene, 6.5 kb in length, contains nine exons with a total length of 2169 nucleotides. Remarkably, the intervening sequences map at positions that fully correspond to those of the vimentin gene. The derived complete primary structure for hamster desmin (468 amino acids; 53,250 daltons) reveals striking species variations in the NH2-terminal domain of desmin. A plasmid containing the complete transcription unit of the desmin gene was transfected into hamster lens cells and into human epithelial (HeLa) cells. In both nonmuscle cell lines the desmin gene was biologically active. The synthesized desmin assembled into authentic IFs, as monitored by immunofluorescence. Double immunofluorescence staining showed that the newly formed desmin filaments colocalize with preexisting vimentin filaments, but not with preexisting keratin filaments.

Chemical and biological aspects of sparsomycin, an antibiotic from Streptomyces.

Publisher Summary This chapter discusses the chemical and biological aspects of sparsomycin, an antibiotic from streptomyces. Sparsomycin is a strong inhibitor of protein biosynthesis, its site of interaction being the large ribosomal subunit, which prevents peptide transfer by interfering with the peptidyl transferase centre. Valuable information on the structure of this peptidyl transferase center and of the ribosomes as a whole has been obtained from studies using sparsomycin. A decision on whether sparsomycin—or its most promising analog—shows sufficiently auspicious activity to warrant further clinical development as an anticancer drug is required. This antibiotic has achieved prominence as a tool to study the protein biosynthesis machinery. A comprehensive review of sparsomycin, its chemistry, biological activity, and potential as an anticancer drug is timely. The compound has attracted much attention since its discovery, not only because of its biological activity but also for its unique structural characteristics and synthetic challenge.

The role of the hydroxymethyl function on the biological activity of the antitumor antibiotic sparsomycin

Abstract The synthesis is described of the sparsomycin analogues11–14 from the l -amino acids valine, isoleucine, phenylalanine and proline, respectively. The sparsomycin derivative 21 was not prepared in a similar way from glycine, but from cystamine following a different reaction route. These analogues, as well as the O-methylated and O-acetylated derivatives 15 and 16, respectively, were tested in vitro for their protein synthesis inhibitory activity and for their inhibition of colony formation of murine leukemia L1210 cells. The results of these assays indicate that the hydroxymethyl function of 1 is not essential for its biological activity, and that increase of lipophilicity in this “northern” region of 1 does not noticeably affect the activity of the drug.

Vimentin and Desmin cDNA Clones: Structural Aspects of Corresponding Proteins and Genes

Microtubules, microfilaments, and intermediate-sized filaments (IF) are the major cytoskeletal elements of most eukaryotic cells. The function of the IF is far from being fully understood. Maintenance of cell shape and/or anchorage to the substratum have been suggested to represent at least some of the functions of these fibrous structures.’ Identification and visualization of the spatial orientation in intact cells have been achieved by electron micro~copy~’~ and immunofluorescence microscopy.4~’ Mainly from the latter studies it appeared that as a rule IF proteins are tissue-specific. For instance mesenchymal cells comprise vimentin as subunit of their IF, whereas cells from an epithelial source contain cytokeratins. An exception of the aforementioned regularity is the vertebrate lens that due to its epithelial origin should produce prekeratin. Nevertheless, only vimentin is found and none of the other IF protein subunits! The reason for deviation from the “rule” is not clear yet. In view of the observation that cells from different embryonic origin start synthesizing vimentin as soon as they are subjected to culturing, it might well be that the eye lens, due to its peculiar growth pattern, arising from a monolayer of epithelial cells that are attached to a natural substratum (the lenticular capsule),’ resembles to some extent cells growing in culture (FIGURE 1). This example of regulation of IF gene expression led us to undertake investigations upon the molecular basis of this process. In order to facilitate comparative studies we performed molecular cloning of both vimentin and desmin cDNAs. The isolated clones served primarily a threefold aim: The determination via the nucleotide sequence of the primary structure of the proteins; the prediction of their secondary structure contained in a general model, presumably valid for all IF protein subunits; and the isolation of the corresponding genes from genomic DNA. Moreover, the isolated genes whose structures have been determined will be used for expression studies in heterologous cell systems.

Pharmacokinetics of the antitumor antibiotic n-pentyl-sparsomycin in beagle dogs

SummaryN-pentyl-sparsomycin (PSm) is a lipophilic analogue of sparsomycin (Sm), which is a well known inhibitor of protein synthesis. This compound was selected for preclinical pharmacokinetic studies because of its high in vitro and in vivo antitumor activity. In this study in which the drug was evaluated in beagle dogs under anaesthesia, the drug concentrations in plasma, urine and bile samples were determined using high performance liquid chromatography (HPLC). Plasma protein binding was approximately 54%. The mean t1/2 β was 0.2 hours (12 minutes) and t1/2 τ was 0.75 ± 0.1 hours (45 ± 6 minutes). During continuous infusions up to 5.25 hours, the steady state was reached in 3 out of 6 experiments, suggesting that in some cases the real t1/2 τ was longer than measured. PSm was actively reabsorbed from the renal tubuli. This process was saturable at the higher doses. Tubular reabsorption played only a minor role in pharmacokinetics as most of the drug (67%) was eliminated by the non-renal clearance. The non-renal clearance was saturable at higher doses of PSm and was the reason for non-linearity of pharmacokinetics.

In vivo antitumor activity of sparsomycin and its analogues in eight murine tumor models.

SummarySparsomycin (Sm) is a known inhibitor of ribosomal protein synthesis with an attractive anticancer potential. Recently, several analogues of Sm which are more active than the parent drug were selected for further study on the basis of in vitro investigations. Six analogues as well as the parent drug were tested for their antitumor activity in eight in vivo murine tumor models: P388 and L1210 leukemias, RC renal cell carcinoma, B16 melanoma, C38 colon carcinoma, LL Lewis lung carcinoma, C22LR osteosarcoma and M5076 sarcoma. Sm itself appeared to have only borderline activity on L1210 leukemia. The analogues that were most active in vitro showed also the highest in vivo activity. The most sensitive tumors were RC, L1210 and P388. Minimal activity was found on B16 and no activity on C22LR, M5076, C38 and LL. The most active compounds are deshydroxy-Sm, ethyl-deshydroxy-Sm and n-pentyl-Sm. There was a considerable loss of activity when L1210 leukemia was implanted sc while the drugs were administered iv. Only one drug, ethyl-deshydroxy-Sm appeared to be active in this assay. No single most effective compound could be found in this study. The overall activity of Sm and its analogues is moderate. The three analogues which show high activity in three ascitic tumors will be further investigated using human tumor xenograft models.

In vivo potentiation of cis-diamminedichloroplatinum (II) antitumor activity by pretreatment with sparsomycin

The influence of protein synthesis inhibition by sparsomycin (Sm) on in vivo cisplatin activity has been studied on BALBc X DBA2: F1 mice bearing L1210 leukemia i.p. Sm alone at the dose range from 0.5 to 3.0 mg/kg did not significantly improve animal survival. Sm potentiated cisplatin activity only when given 3 or 6 h prior to cisplatin (P less than 0.001). Sm 0.5-1.5 mg/kg 3 h prior to cisplatin resulted in a significant prolongation of animal survival (P less than 0.001) and 66% cures in each group versus 0% due to cisplatin alone. Sm pretreatment decreased weight loss due to cisplatin suggesting that it probably is able to decrease cisplatin toxicity.

Pharmacokinetics and toxicology of sparsomycin in beagle dogs

SummarySparsomycin is a cytotoxic drug exhibiting a broad spectrum of in vitro activity against murine tumors and many tumor cell lines. It also appears to be a potent stimulator of the antitumor activity of cisplatin against L1210 leukemia in vivo. However, because of its toxicity, the antitumor activity of sparsomycin on murine tumors in vivo has been disappointing. The purpose of our study was to investigate the pharmacokinetics of this drug as well as the possible mechanisms that produce sparsomycin toxicity. Tests on beagle dogs revealed that about 60% of the drug is eliminated by metabolic clearance, while 40% is eliminated by the kidneys. After a single bolus injection of 0.1 mg/kg sparsomycin without narcosis, sparsomycin was eliminated with a tβ1/2 of 0.6–0.7 h, the AUC being 0.32–0.38 mg·h·1-1, and the volume of distribution (Vd) 0.26 l/kg. In addition to being subject to glomerular filtration, sparsomycin is probably also actively excreted and actively reabsorbed by the renal tubuli. Sparsomycin itself may inhibit its active tubular excretion, thus resulting in a decrease in the drugs renal clearance and its accumulation in the plasma. Sparsomycin appeared to be toxic primarily in the liver, disturbing its function and the synthesis of plasma proteins. Two out of five dogs developed hemorrhagic diathesis due to hypofibrinogenemia and deficiency of other blood-coagulation factors. Sparsomycin was not toxic to the bone marrow.