Stringner S. Yang

Physicochemical analysis of the deoxyribonucleic acid product of murine intracisternal A particle RNA-directed DNA polymerase.

The nascent DNA transcript of intracisternal A particle RNA-directed DNA polymerase appeared to be covalently linked to an RNA primer. Fidelity of transcription is evident since the DNA transcript hybridized specifically back to 35-70 S RNA of intracisternal A particles but not with heterologous RNAs. This DNA transcript has an approximate molecular weight of 145 000, estimating 350 polynucleotides and base ratios with an excess of dGMP.

Intracisternal a particle-specific dna sequences in mammary tumor cells, hybrids, and cybrids derived from laboratory mice and from feral mice of mus musculus and mus cervicolor.

Abstract We have examined the DNA sequences homologous to murine intracisternal A-particle (IAP) cDNA by nucleic acid hybridization, using mammary tumor (MT) cells, hybrids and cybrids, and feral mouse cells of Mus musculus and Mus cervicolor . The radioactive cDNA probe was synthesized by the endogenous reverse transcriptase reaction in purified mammary tumor IAP. Given the copy numbers of IAP-specific DNA sequences obtained in the various parent cells, hybrids, and cybrids, and the thermal stability characteristics of the DNA duplexes, two distinct types of phenomena were observed. First, one phenotypically negative recipient cell line, 3T3-4EF, had only 1–2 copies of IAP-specific DNA sequences per haploid genome prior to fusion. When this line was fused to MT cytoplasts (enucleated cells) containing IAP, the resulting cybrid cells were found to have about 10 copies of IAP-specific DNA sequences, which by the Hirt procedure, localized in the detergent-insoluble fraction of the nuclear DNA. Thus the continuous replication of IAP in the MT x 3T3-4EF cybrids was associated with an increase in the number of IAP-specific DNA sequences in the cell genome. Second, another phenotypically negative recipient cell line, INC, had about 4 copies of IAP-specific DNA sequences per haploid genome prior to fusion. Following fusion with MT cytoplasts, the resulting MT x INC cybrids did not show any increase in the number of IAP-specific DNA sequences, even after IAP replication had become overt, continuous, and nonsegregated. The distribution of DNA copies in the various Mus musculus and Mus cervicolor laboratory and feral mouse cells indicates that the IAP genome has been conserved in spite of the divergency of the two species.

The Camptothecin Experience: From Chinese Medicinal Plants to Potent Anti-Cancer Drugs

Camptothecin is a potent natural product based anticancer agent isolated from an organic extract of the bark of a Chinese tree, Camptotheca acuminata, Decaisne (Nyssaceae). The chemical identification of camptothecin was first reported in 1966 by Wall et al (1) as a plant alkaloid which showed strong potency against a number of cancer cells derived from leukemia, small cell lung cancer, and colon and rectal cancer. This communication describes the drug discovery and development process of camptothecin from Camptotheca acuminata at the NCI in collaboration with Monroe E. Wall’s group of the Research Triangle Institute and other laboratories facilitated by NCI support. This led to the eventual development of several potent camptothecin analogs, 9-aminocamptothecin, topotecan, irinotecan, and rubitecan, which have been approved for cancer therapy against diverse cancer types including different leukemias and lymphoma, small cell lung carcinoma, colon and rectal cancer, cancer of the central nervous system, renal cell carcinoma, ovarian and breast cancer in humans. Topotecan and irinotecan are also currently used in a wide spectrum of clinical trials in patients with metastatic cancers. The resolution of camptothecin action mechanism targeting topoisomerase I provided vital information in cancer drug action, and stimulated novel and rational cancer drug design and development.

Activation and Specificity of Aflatoxin B 1 Binding on hhc M , a Human Oncogene of Low Efficiency, and Cell Transformation

Aflatoxin B1 (AFB1), a metabolite of Aspergillus flavus, chemically classified as a furocoumarin, is known to be the most potent hepatocarcinogen experimentally tested in various species including trout, rat, hamster, dog and rhesus monkey.13,21 The carcinogenic effect resides in the mutagenic potential of AFB1 upon metabolic conversion into the epoxide form by liver mixed function oxidases. By mode of nucleophilic attack, AFB1-epoxide binds to DNA forming covalent bonds with the N7 of deoxyguanine (dG), thus inducing an erroneous base-pairing with deoxyadenine (dA) instead of deoxycytidine (dC) (Fig. 1). In eukaryotes, such as hepatocytes, a strong DNA repair enzyme system usually safeguards against such mistakes by excising the mis-paired base, and an organism depends on continual repair of DNA (Fig. 2). If this erroneous base-pairing eluded the cell’s DNA repair enzyme, at subsequent DNA replication a conversion of dG.dC --> T.dA ensures, thus resulting in a point-mutation.7 Such a mutation generates an irreversible DNA lesion (Fig. 2) and if it is of survival value, it usually produces an altered gene product. On the other hand, a disruption of the open reading frame results if the DNA repairing process excised the mis-paired base without replacement with the correct base, i.e. generated a deletion mutation, or if the T.dA substitution of dG.dC generated a nonsense, i.e. stop codon. This usually results in a lethal mutation.