amanitins and Multiple-Myeloma

Despite major treatment advances in recent years, patients with multiple myeloma inevitably relapse. The RNA polymerase II complex has been identified as a promising therapeutic target in both proliferating and dormant cancer cells. Alpha-amanitin, a toxin so far without clinical application due to high liver toxicity, specifically inhibits this complex. Here, we describe the development of HDP-101, an anti-B-cell maturation antigen (BCMA) antibody conjugated with an amanitin derivative. HDP-101 displayed high efficacy against both proliferating and resting myeloma cells

Topics: Amanitins; Animals; Cell Death; Cell Proliferation; Disease Models, Animal; Enzyme Inhibitors; Female; Humans; Immunoconjugates; Mice; Mice, SCID; Multiple Myeloma

To investigate the molecular mechanisms of action of the nitrogen mustard melphalan in patients treated for multiple myeloma, the in vivo induction and repair of melphalan-induced DNA damage was measured in genes with different transcriptional activity (b-actin>p53>N-ras>d-globin) from leukocytes of 20 multiple myeloma patients following chemotherapeutic administration of high-dose melphalan (200mg/m(2)) and autologous blood stem cell transplantation. Heterogeneous repair was found among the studied genes. The extent of repair was always in the order: b-actin>p53>N-ras>d-globin, correlating with the gene transcriptional state. Similar findings were obtained using peripheral blood mononuclear cells (PBMC) from healthy volunteers following in vitro treatment with melphalan, indicating that these results are not malignant disease-specific. Following in vitro treatment of PBMC from healthy volunteers with alpha-amanitin, an inhibitor of RNA polymerase II that can also induce condensation of chromatin structure, a significant inhibition of the removal of melphalan-induced damage in the three active genes but not in the silent d-globin gene was found, suggesting that transcription and/or chromatin structure may play important roles in the preferential DNA repair. When the in vivo DNA damage formation and repair in multiple myeloma patients following chemotherapeutic administration of melphalan was measured in the two strands of the active genes, no strand bias was found, indicating that the global genome repair subpathway of nucleotide excision repair may play a crucial role in the repair of these adducts. These results were also confirmed in PBMC from healthy volunteers following in vitro treatment with melphalan. Using micrococcal nuclease digestion of nuclei isolated from PBMC of multiple myeloma patients before the chemotherapeutic treatment, as well as from PBMC of healthy volunteers, we probed the chromatin structure in each gene and found that the "looseness" of the chromatin structure correlated with the levels of the gene-specific repair, being again in the order: b-actin>p53>N-ras>d-globin. To conclude, the in vivo gene-specific repair of melphalan-induced damage in humans is greatly affected by the local chromatin structure.

Topics: Actins; Amanitins; Antineoplastic Agents, Alkylating; Chromatin; DNA Damage; DNA Primers; DNA Repair; Genes, p53; Genes, ras; Humans; Leukocytes, Mononuclear; Melphalan; Molecular Probe Techniques; Multiple Myeloma; Plasmids

The transcription of U1 RNA genes was studied in isolated nuclei from mouse myeloma cells. Using a cloned U1b gene as a probe, we showed that isolated nuclei synthesize both U1b and U1a RNA. The U1 RNAs were initiated in vitro, as measured by incorporation of adenosine 5'-O-(2-thiotriphosphate) into U1 RNA. There was transcription of the 3'-flanking region but no transcription of regions directly 5' to the U1 genes. In addition to U1 RNAs of the correct length which were released from the nuclei, there were larger RNAs, presumably resulting from transcription into the 3'-flanking region, which were retained in the nuclei. Chase experiments showed that these longer transcripts were not precursors to mature U1 RNA, a finding consistent with the idea that 3'-end formation is coincident with transcription. During the chase, there was maturation of the 3' ends of U1a and U1b RNAs from slightly longer precursors. In addition to accurate transcription of U1 RNA, there was also synthesis of U2 and U3 RNA. All three of these RNAs were transcribed by RNA polymerase II, as measured by their sensitivity to alpha-amanitin.

Topics: Adenosine Triphosphate; Amanitins; Animals; Cell Nucleus; Cell-Free System; DNA, Recombinant; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Mice; Multiple Myeloma; Nucleic Acid Hybridization; RNA, Small Nuclear; Thionucleotides; Transcription, Genetic; Tumor Cells, Cultured

Topics: Amanitins; Animals; Carcinoma 256, Walker; Carcinoma, Hepatocellular; DNA; DNA-Directed RNA Polymerases; Kidney; Kinetics; Liver Neoplasms; Magnesium; Manganese; Multiple Myeloma; Neoplasms, Experimental; Organ Specificity; Rats; Sarcoma, Yoshida; Spleen

Nuclei isolated from mouse myeloma cells grown in tissue culture are capable of synthesizing RNA for prolonged periods of time. Addition of cytoplasmic extracts to the system stimulates slightly the rate and prolongs the time of synthesis. As judges by sedimentation in SDS and in formamide gradients, the size of the RNA synthesized is heterogeneous from smaller than 10S to larger than 45S, thus resembling in vivo made RNA. The characteristics of some of the RNA are in keeping with those expected to be for mRNA. Fifty percent of the RNA synthesis is sensitive to alpha-amanitin. After an incubation of two hours in the absence of alpha-amanitin about 10 percent of the newly synthesized RNA is found outside of the nuclei; it sediments with a broad distribution at 18S. A considerable fraction of the RNA that is released from nuclei in vitro can promote the formation of polyribosomes, and contains molecules that are polyadenylated and "capped".

Topics: Amanitins; Animals; Cell Nucleus; Cells, Cultured; Cytoplasm; Guanosine Triphosphate; Kinetics; Mice; Molecular Weight; Multiple Myeloma; Poly A; Polyribosomes; Reticulocytes; Ribonucleases; RNA, Messenger; RNA, Neoplasm; Uridine