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This paper introduces a series of invited essays on current controversies in basic cancer research. The initial group of essays focuses on the detection and interpretation of molecular and cellular changes suspected to be of importance in the cause and pathogenesis of cancer. There are two formats: (i) differing viewpoints are presented in parallel, or (ii) the author(s) evaluate a hypothesis in light of available data. Each type of paper aims to critically evaluate current hypotheses and supporting data, while avoiding pronouncements on validity. Recent advances in molecular biology now permit us to consider genes as chemical entities. Individual genes can be isolated, cloned to produce multiple copies, sequenced, and assayed for biological function. This new molecular technology is being applied to fundamental questions in cancer research. The controversies resulting from these pioneering studies are the topics of the initial papers in this series. Forthcoming essays will concern the mechanism(s) of tumor promotion; a search for cancer genes by DNA transfection; the role of DNA rearrangements as initiating events in carcinogenesis; the O6 position of guanine as a critical target of carcinogens; and metals as mutagens and carcinogens.

We have measured the forward mutation rate at the hypoxanthine phosphoribosyltransferase (HPRT) gene of the human promyelocytic leukemia cell line HL-60 and have determined the molecular spectrum of spontaneous HPRT mutations in 45 independent 6-thioguanine-resistant HL-60 sublines. Four fluctuation tests using a total of 132 replicate HL-60 cultures revealed a mean forward mutation rate of HL-60 cells to thioguanine resistance of 1.7-6 x 10(-7)/cell/generation. Blot hybridization analysis of the X-linked HPRT gene using a human HPRT complementary DNA probe revealed abnormalities in HPRT gene structure and/or HPRT mRNA expression in 24 of 45 (53%) independent thioguanine-resistant HL-60 sublines. Six different classes of mutation were identified. The most prevalent (47%; 21 of 45 mutations) consists of mutations that are not detected by blot hybridization analyses and that do not disrupt HPRT mRNA production. These results suggest that a comparatively low forward mutation rate may be found in malignant human cells that exhibit both karyotypic and molecular evidence of genomic instability and that several different molecular classes of mutation may contribute to thioguanine resistance in HL-60, and perhaps in other, malignant human cells. The forward mutation assay system we have developed using the X-linked HPRT gene of HL-60 cells may be useful for analyses of the mutagenic potential and molecular spectrum of mutations produced by chemotherapeutic agents, suspected human mutagens and carcinogens, and phagocyte respiratory burst oxidants in human cells.

The association of aluminum dusts and pulmonary fibrosis with emphysema in workers in the aluminum processing and manufacturing industries is well established. The early and minimal reactions of the lungs to the aluminum dusts are not known. This report presents the first case of pulmonary granulomatosis associated with aluminum inhalation. The occupational history of this patient was thoroughly examined, and the aluminum was identified by electron probe microanalysis of the lung biopsy specimen. The granulomatous response in this patient was similar to that observed in rabbits following aluminum dust inhalation. Hypersensitivity or individual idiosyncrasy may play a role in the development of the pulmonary granulomas following exposure to aluminum.

Recombinant DNA techniques have been used to quantitate the amount of nucleotide sequence divergence in the mitochondrial DNA population of individual normal humans. Mitochondrial DNA was isolated from the peripheral blood lymphocytes of five normal humans and cloned in M13 mp11; 49 kilobases of nucleotide sequence information was obtained from 248 independently isolated clones from the five normal donors. Both between- and within-individual differences were identified. Between-individual differences were identified in approximately 1/200 nucleotides. In contrast, only one within-individual difference was identified in 49 kilobases of nucleotide sequence information. This high degree of mitochondrial nucleotide sequence homogeneity in human somatic cells is in marked contrast to the rapid evolutionary divergence of human mitochondrial DNA and suggests the existence of mechanisms for the concerted preservation of mammalian mitochondrial DNA sequences in single organisms.

Thioguanine-resistant primary clones were grown from single cell suspensions obtained from dog and human kidneys by enzymatic digestion. In medium containing a relatively high concentration (10 micrograms/ml) of thioguanine, thioguanine-resistant primary clones arose from each source at frequencies ranging from 10(-4) to 10(-5). A reduction in total hypoxanthine uptake was found in the thioguanine-resistant primary clones which had developed in thioguanine medium, consistent with a reduction in hypoxanthine phosphoribosyltransferase activity. When these thioguanine-resistant primary clones were subsequently grown in the absence of thioguanine and assayed for the thioguanine-resistant phenotype and hypoxanthine phosphoribosyltransferase activity, it was found that most were now thioguanine-sensitive and yielded cell-free extracts with substantial amounts of hypoxanthine phosphoribosyltransferase activity. In contrast, thioguanine-resistant human clones grown continuously in the presence of thioguanine yielded cell-free extracts with little or no detectable hypoxanthine phosphoribosyltransferase activity. Southern blot analysis demonstrated no structural alterations in the hypoxanthine phosphoribosyltransferase gene in thioguanine-resistant primary human kidney clones. These results suggest that a novel mechanism(s) for thioguanine resistance and the control of hypoxanthine phosphoribosyltransferase expression may occur in dog and human kidney cells.

Recombinant DNA techniques have been used to search for mitochondrial (mt) nucleotide (nt) sequence differences between human tissues within an individual. mtDNA isolated from brain, heart, liver, kidney, and skeletal muscle of two different individuals was cleaved with SacI and XbaI, and then cloned in bacteriophage M13. Partial nt sequence determination of 121 independently isolated recombinant M13 clones containing either the cytochrome oxidase subunit III gene or the D-loop region of human mtDNA revealed base substitution differences between individuals, and between each individual and the published human mtDNA sequence. A majority of these base substitutions were transitions. No systematic nt sequence differences were identified between tissues within an individual, however. These results suggest that mtDNA sequence alterations do not accompany organogenesis, and that somatic mutations do not accumulate in the mtDNA of different human tissues to a level of greater than one nt substitution per molecule.

Several regions of the human mitochondrial genome are refractory to cloning in plasmid and bacteriophage DNA vectors. For example, recovery of recombinant M13 clones containing a 462 basepair MboI-Kpn I restriction fragment that spans nucleotide positions 15591 to 16053 of HeLa cell mitochondrial DNA was as much as 100-fold lower than the recovery of M13 clones containing other regions of the human mitochondrial genome. All of 50 recombinant M13 clones containing this 'uncloneable' fragment had one or more changes in nucleotide sequence. Each clone contained at least one alteration in two nucleotide positions within the tRNAThr gene that encode portions of the anticodon loop and D-stem of the HeLa mitochondrial tRNAThr. These results imply that the HeLa mitochondrial tRNAThr gene is responsible for the 'uncloneable' phenotype of this region of human mitochondrial (mt) DNA. A total of 61 nucleotide sequence alterations were identified in 50 independent clones containing the HeLa mt tRNAThr gene. 56 mutations were single-base substitutions; 5 were deletions. Approximately 80% of the base substitution mutations were A:T----G:C transitions. A preference for A:T----G:C transition mutations also characterizes polymorphic base substitution variants in the mitochondrial DNA of unrelated individuals. This similarity suggests that human mitochondrial DNA sequence variation within and between individuals may have a common origin.

Werner syndrome (WS) is a rare autosomal-recessive disorder characterized by the premature appearance of features of normal aging in young adults. The extensive phenotypic overlap between WS and normal aging suggests they may also share pathogenetic mechanisms. We reported previously that somatic cells from WS patients demonstrate a propensity to develop chromosomal aberrations, including translocations, inversions, and deletions, and that WS cell lines demonstrate a high spontaneous mutation rate to 6-thioguanine resistance. We report here the biochemical and molecular characterization of spontaneous mutations at the X chromosome-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in 6-thioguanine-resistant WS and control cells. Blot hybridization analysis of 89 independent spontaneous HPRT mutations in WS and control mutants lacking HPRT activity revealed an unusually high proportion of HPRT deletions in WS as compared with control cells (76% vs. 39%). Approximately half (58%) of the deletions in WS cells consisted of the loss of greater than 20 kilobases of DNA from the HPRT gene. These results suggest that an elevated somatic mutation rate, and particularly deletions, may play pathogenetically important roles in WS and in several associated age-dependent human disease processes.

The mutagenic potentials of ethylmethane sulfonate, N-methyl-N'-nitrosoguanidine, and benzo(a)pyrene diol-epoxide in human mitochondria were determined by cloning and nucleotide sequencing of mitochondrial (mt) DNA from HeLa cells treated with these mutagens. Mutagen concentrations that reduced cell survival to approximately 0.1% of untreated cultures were used. Mitochondrial DNA was prepared 2 to 3 weeks after mutagen treatment, at which time the treated cell population had regrown to 10 times the starting cell number. In one series of experiments, a portion of the D-loop region of mtDNA from treated or control HeLa cells was cloned into the bacteriophage vector M13mp19, and the nucleotide sequences of 102 independent clones were determined. Only a single G:C base pair deletion was observed in 1 of 12 clones derived from HeLa cells treated 6 times with ethylmethane sulfonate. From benzo(a)pyrene diol-epoxide-treated HeLa cells, G:C base pair deletions were found in 14 of 63 clones. All 14 of these G:C deletion mutations occurred at the same position in independent clones, however, and thus could be the progeny of a single mutational event. In a second series of experiments, a method for the selection of mtDNA mutants was utilized. Mutations in an "uncloneable" fragment of human mtDNA render the fragment cloneable and thus provide a selection for mutations in this region of human mtDNA. No enhancement in the cloning efficiency of this region of mtDNA was observed after exposure of cells to toxic concentrations of either MNNG or benzo(a)pyrene diol-epoxide. Moreover, the site and types of nucleotide sequence alterations observed after mutagen treatment were similar to those obtained in the absence of drug treatment. The results of both types of experiments suggest that mutagenesis of human mtDNA is an infrequent event, even after extensive treatment of HeLa cells with potent mutagens that can covalently modify mtDNA.

I have provided a brief overview of our experience teaching undergraduate general pathology at the University of Washington School of Medicine. Our course is part of a pathology curriculum that is somewhat unusual in light of the amount of time we devote to general, as opposed to organ system pathology. We think this makes sense in relation to the way medical teaching and practice are changing. Resources and curriculum time needed to teach an extensive, morphology-based organ system pathology curriculum are no longer available. In addition, experimental biology and medicine are beginning to improve the way human diseases are diagnosed and treated. Many of these advances are the result of new information on disease aetiology and pathogenesis. Students and practitioners of medicine need an understanding of disease processes that will allow them to rapidly assimilate and rationally apply this new information. The particular strengths of our course, as we view them, are an opportunity to discuss the small number of processes that underlie most human disease in some depth, and thus to emphasize general pathology as a conceptual and practical foundation for the practice of medicine; our laboratory sessions, which in a sense illustrate and summarize the course; and early placement of the course in the curriculum, which allows us to capitalize on concurrent basic science courses and a high level of interest among students in applying basic science knowledge to understanding human disease. Problems we face include the need for more 'active' learning exercises in the lecture and laboratory format we are bound to; the need, given the scope of general pathology, to present more 'take home messages' and fewer systematic reviews of evidence than we would like; the limited clinical knowledge of first year students, which restricts the scope of our laboratory and disease example presentations; and an inability to consistently challenge the abilities of the best students in each class. Organizing and teaching the course described above has been in large part satisfying and stimulating. I hope this overview has provided useful ideas for others teaching or contemplating courses in general pathology.

Nucleotide sequence variation in mitochondrial DNA isolated from human leukemic cells has been analyzed by recombinant DNA techniques. Three hundred eighty-seven independent recombinant DNA clones, each containing one of three defined segments of mitochondrial DNA isolated from the neoplastic cells of four leukemic patients, were analyzed. Partial nucleotide sequence determination of the 387 clones yielded a total of 81.7 kilobases of nucleotide sequence information. The only evidence of within-individual nucleotide sequence divergence consisted of three clones containing deletions of one or two nucleotides in one mitochondrial DNA region. These clones were three of 113 independent clones isolated from a patient with acute lymphocytic leukemia. The low level of nucleotide sequence divergence in the mitochondrial DNA population of neoplastic cells from individual leukemic patients suggests that a mechanism or mechanisms exist that limit the development of nucleotide sequence divergence in mammalian mitochondrial DNA. The results further suggest that this mechanism does not appear to be abrogated by neoplastic transformation in leukemic patients.