Eberhard Amtmann

The simultaneous extraction of high-molecular-weight DNA and of RNA from solid tumors

A novel method for the isolation of both macromolecular DNA and RNA from solid tissues based upon the disruption by vibration of deep-frozen material in a mechanical device termed Mikro-dismembrator, is described. This technique reveals a yield of, on the average, 1 to 3 mg of either DNA or RNA per gram of tissue. The quality of the purified nucleic acids permits the detailed analysis of integrated tumor virus DNA sequences and their mRNA transcripts. Furthermore, the efficient isolation of papilloma virions from keratinized wart tissue is facilitated by the application of the Mikro-dismembrator.

Lauric acid inhibits the maturation of vesicular stomatitis virus

In the presence of lauric acid (C12), the production of infectious vesicular stomatitis virus (VSV) was inhibited in a dose-dependent manner. The inhibitory effect was reversible; after removal of C12 the antiviral effect disappeared. In addition, the chain length of the monocarboxylic acids proved to be crucial, as those with shorter or longer chains were less effective or had no antiviral activity. Concomitant with the C12-induced inhibition was the stimulation of triacylglycerol synthesis, increasing the amount up to ninefold. Analysis of the antiviral mechanism of C12 revealed that the correct assembly of the viral components was disturbed, but viral RNA and protein synthesis remained unimpaired. By cell fractionation and Western blot analysis the amount of viral M protein located in the plasma membrane was found to be markedly reduced after treatment with C12, whereas in the cytoplasm the quantity of M protein was similar to that in untreated cells. C12 did not influence M protein synthesis, but prevented the binding of M protein to the host cell membrane, where the protein plays an essential role in virus assembly. Thus, treatment of VSV-infected cells with C12 resulted in inhibition of virus release. It is suggested that the newly synthesized triacylglycerols might interact with the host cell plasma membrane and interfere with virus maturation.

Activation of latent papillomavirus genomes by chronic mechanical irritation.

The skin of animals of a laboratory strain of Mastomys natalensis carrying endogenous, latent papillomavirus genomes was irritated by scratching with glasspaper. Hyperproliferation of the epidermis and amplification of viral DNA followed this treatment, and in approximately 27% of the animals virus-producing papillomas were induced.

Selective killing of tumor cells by xanthates

Xanthate derivatives of primary alcohols with antiviral properties exert, in combination with monocarboxylic C11 or C12 acids a pronounced anti-tumor activity in vitro and in vivo. Tricyclodecan-9-yl-xanthogenate (D609) or cyclododecyl xanthogenate (D435) when administered together with either undecanoic or dodecanoic acid to various transformed animal and human tumor cells (displaying low serum requirement) cause cell death. In contrast, normal cells from which transformed derivatives arose, were unaffected.

Interruption of growth signal transduction by an antiviral and antitumoral xanthate compound

The binding of growth factors to the cellular receptors elicits the phosphorylation of proteins which transmit growth signals to the nucleus [E. Rozengurt (1986) Science 234, 161-166]. Both the tyrosine-specific kinase (growth factor receptor) and the threonine-serine phosphorylating protein kinase C (pkC) become activated upon binding of the epidermal growth factor (EGF) to its receptor. Here we describe the selective inhibition of the pkC activation by tricyclodecane-9-yl-xanthogenate (D609) in the presence of unsuppressed receptor tyrosine autophosphorylation. As a consequence the affinity of EGF to the receptor was not down-regulated and the complex failed to be internalized.

Inhibition of the phosphorylation of the regulatory non-structural protein of vesicular stomatitis virus by an antiviral xanthate compound.

The growth of vesicular stomatitis virus (VSV) can be inhibited by the antiviral compound tricyclo-decane-9-yl-xanthogenate (D609). On analysing the antiviral mechanism we found no effect on the primary transcription of infecting VSV genomes. In contrast, the processes of replication and transcription during late stages of infection were inhibited. Despite the synthesis of all five virus-coded proteins (41% to 56% of the uninhibited control), as shown by labelling with [35S]methionine, the phosphorylation of the non-structural (NS) protein was reduced in the presence of the xanthate by a factor of at least 17. The pattern of phosphorylation of the bulk of cellular proteins remained unaltered under the same conditions. A relation between a possible loss of biological activity of the NS protein owing to the lack of phosphorylation and the decreased VSV RNA synthesis is suggested.

Synergistic antiviral effect of xanthates and ionic detergents

Xanthate compounds have been shown to exhibit antiviral activity against various DNA and RNA viruses under acidic pH conditions. It is now possible to utilize the unique broad range antiviral spectrum of these compounds under physiological pH conditions (pH 7.4) by simultaneous administration of certain ionic detergents. When used in conjunction with tricyclodecan-9-yl-xanthate (D609), sodium deoxycholate, sodium dodecylsulfate and certain fatty acids, which have no antiviral activity of their own, inhibit the replication of various DNA and RNA viruses (such as herpes simplex, vesicular stomatitis and Coxsackie B 4) in vitro at pH 7.4. Among saturated fatty acids of various chain lengths there was a marked size restriction in that the efficiency of undecanoic acid (11 C atoms) was three orders of magnitude greater than that of shorter (6 C atoms) or longer (18 C atoms) monocarbonic acids. Dose-response kinetics revealed a synergistic interaction between the xanthate and the monocarbonic acid. A dose that inhibited the replication of herpesvirus by a factor of 1000 still permitted mitotic activity in uninfected growing control cultures.

Systemic treatment of a human epidermoid non-small cell lung carcinoma xenograft with a xanthate compound causes extensive intratumoral necrosis.

Therapeutic effects were obtained after systemic treatment of athymic mice bearing an epidermoid non-small cell human lung carcinoma (NSCLC) xenograft with tricyclodecan-9-yl xanthogenate (D609) and the potassium salt of a fatty (dodecanoic) acid. Extensive intratumoral necrosis was observed 3 days after the treatment.

Antitumoral activity of a xanthate compound I. Cytotoxicity studies with neoplastic cell lines in vitro

Xanthate derivatives were shown previously to display antitumor activity against transformed fibroblasts and lymphoma cells in combination with monocarboxylic acids [1]. Various malignant cell lines of human origin were treated in vitro to explore the range of antitumoral activity of the compounds. The combination of tricyclodecan-9-yl-xanthogenate (D 609) with undecanoic acid (C11) exerted dose dependent cytotoxic and antiproliferative effects on cell lines both from solid tumors (glioblastomas, colon-carcinomas) and hematological diseases (lymphomas, CML/BC). Additionally, the combination of D 609/C11 was able to kill both methotrexate- and adriamycin-resistant L 1210 and S 180 cells, indicating that there is no cross-resistance for these drugs and D 609/C11 in vitro.

Interaction between latent papovavirus genomes and the tumor promoter TPA

The tumor promoting agent 12-O-tetradecanoylphorbol-13acetate (TPA) enhances both the transformation of rat embryo cells by adenovirus and the cloning efficiency of such cells [ 11. Induction of Epstein-Barr virus (EBV) antigens and stimulation of particle synthesis by treatment of the EBV-carrying cell lines with TPA was reported in [ 2,3]. Here we report that TPA exclusively enhances the viral DNA content of cells which harbor papovavirus genomes in a latent episomal state. On the other hand, no effect is exerted by TPA on the copy number of the viral DNA in productively infected cells and on integrated papovavirus DNA in transformed cells.