CHROMOSOMAL ORIGIN OF DM-DNA

Double minutes (dm) are small chromatin particles of 0.3 microns diameter found only in the metaphase cells of human and murine tumors. Dm are unique cytogenetic structures since their numbers per cell show wide variation. At cell division, dm are retained despite the lack of centromeres. In squash preparations, dm show clustering often in association with chromosomes. Human carcinoma cell line SW613-S18 was found to have large numbers of dm and biological characteristics favorable for mitotic synchronization and chromosome isolation experiments.^ S18 cells were synchronized to mitosis with metabolic and mitotic blocking compounds. Mitotic cells were lysed to release chromosomes and dm from the mitotic spindle and the resulting suspensions were fractionated to enrich for dm. The DNA in enriched fractions was characterized. The reassociation kinetics of dm-DNA driven with placental human DNA was similar to the reassociation curve of labeled placental DNA under similar conditions. In situ hybridization of dm-DNA to tumor and normal metaphase cells showed grain localization over the entire karyotype. Dm-DNA was shown by pulse chase DNA replication experiments to replicate during early and mid S-phase of the cell cycle, but not in late S-phase. In addition, BrdUrd incorporation studies showed that dm-DNA replicates only once during the S-phase. Premature chromosome condensation studies suggest the basis of numerical heterogeneity of dm is nondisjunction, not anomalous or unscheduled DNA replication.^ These data and previous cytochemical banding studies of dm in SW613-S18 indicate that dm-DNA is chromosomal in origin. No evidence of gene amplification was found in the DNA reassociation data. It is likely that dm-DNA represents the pale-staining G-band regions of the human karyotype in this cell line. ^

CYTOGENETIC AND DNA CONTENT ANALYSIS IN MULTIPLE MYELOMA

In this dissertation, the cytogenetic characteristics of bone marrow cells from 41 multiple myeloma patients were investigated. These cytogenetic data were correlated with the total DNA content as measured by flow cytometry. Both the cytogenetic information and DNA content were then correlated with clinical data to determine if diagnosis and prognosis of multiple myeloma could be improved.^ One hundred percent of the patients demonstrated abnormal chromosome numbers per metaphase. The average chromosome number per metaphase ranged from 42 to 49.9, with a mean of 44.99. The percent hypodiploidy ranged from 0-100% and the percent hyperdiploidy from 0-53%. Detailed cytogenetic analyses were very difficult to perform because of the paucity of mitotic figures and the poor chromosome morphology. Thus, detailed chromosome banding analysis on these patients was impossible.^ Thirty seven percent of the patients had normal total DNA content, whereas 63% had abnormal amounts of DNA (one patient with less than normal amounts and 25 patients with greater than normal amounts of DNA).^ Several clinical parameters were used in the statistical analyses: tumor burden, patient status at biopsy, patient response status, past therapy, type of treatment and percent plasma cells. Only among these clinical parameters were any statistically significant correlations found: pretreatment tumor burden versus patient response, patient biopsy status versus patient response and past therapy versus patient response.^ No correlations were found between percent hypodiploid, diploid, hyperdiploid or DNA content, and the patient response status, nor were any found between those patients with: (a) normal plasma cells, low pretreatment tumor mass burden and more than 50% of the analyzed metaphases with 46 chromosomes; (b) normal amounts of DNA, low pretreatment tumor mass burden and more than 50% of the metaphases with 46 chromosomes; (c) normal amounts of DNA and normal quantities of plasma cells; (d) abnormal amounts of DNA, abnormal amounts of plasma cells, high pretreatment tumor mass burden and less than 50% of the metaphases with 46 chromosomes.^ Technical drawbacks of both cytogenetic and DNA content analysis in these multiple myeloma patients are discussed along with the lack of correlations between DNA content and chromosome number. Refined chromosome banding analysis awaits technical improvements before we can understand which chromosome material (if any) makes up the "extra" amounts of DNA in these patients. None of the correlations tested can be used as diagnostic or prognostic aids for multiple myeloma. ^

VERIFICATION OF DNA PREDICTED PROTEIN SEQUENCES BY ENZYME HYDROLYSIS AND MASS SPECTROMETRIC ANALYSIS

The focus of this thesis lies in the development of a sensitive method for the analysis of protein primary structure which can be easily used to confirm the DNA sequence of a protein's gene and determine the modifications which are made after translation. This technique involves the use of dipeptidyl aminopeptidase (DAP) and dipeptidyl carboxypeptidase (DCP) to hydrolyze the protein and the mass spectrometric analysis of the dipeptide products.^ Dipeptidyl carboxypeptidase was purified from human lung tissue and characterized with respect to its proteolytic activity. The results showed that the enzyme has a relatively unrestricted specificity, making it useful for the analysis of the C-terminal of proteins. Most of the dipeptide products were identified using gas chromatography/mass spectrometry (GC/MS). In order to analyze the peptides not hydrolyzed by DCP and DAP, as well as the dipeptides not identified by GC/MS, a FAB ion source was installed on a quadrupole mass spectrometer and its performance evaluated with a variety of compounds.^ Using these techniques, the sequences of the N-terminal and C-terminal regions and seven fragments of bacteriophage P22 tail protein have been verified. All of the dipeptides identified in these analysis were in the same DNA reading frame, thus ruling out the possibility of a single base being inserted or deleted from the DNA sequence. The verification of small sequences throughout the protein sequence also indicates that no large portions of the protein have been removed after translation. ^

MATHEMATICAL STUDIES ON THE EVOLUTIONARY CHANGE OF DNA SEQUENCES

With the aim of understanding the mechanism of molecular evolution, mathematical problems on the evolutionary change of DNA sequences are studied. The problems studied and the results obtained are as follows: (1) Estimation of evolutionary distance between nucleotide sequences. Studying the pattern of nucleotide substitution for the case of unequal substitution rates, a new mathematical formula for estimating the average number of nucleotide substitutions per site between two homologous DNA sequences is developed. It is shown that this formula has a wider applicability than currently available formulae. A statistical method for estimating the number of nucleotide changes due to deletion and insertion is also developed. (2) Biases of the estimates of nucleotide substitutions obtained by the restriction enzyme method. The deviation of the estimate of nucleotide substitutions obtained by the restriction enzyme method from the true value is investigated theoretically. It is shown that the amount of the deviation depends on the nucleotides in the recognition sequence of the restriction enzyme used, unequal rates of substitution among different nucleotides, and nucleotide frequences, but the primary factor is the unequal rates of nucleotide substitution. When many different kinds of enzymes are used, however, the amount of average deviation is generally small. (3) Distribution of restriction fragment lengths. To see the effect of undetectable restriction fragments and fragment differences on the estimate of nucleotide differences, the theoretical distribution of fragment lengths is studied. This distribution depends on the type of restriction enzymes used as well as on the relative frequencies of four nucleotides. It is shown that undetectability of small fragments or fragment differences gives a serious underestimate of nucleotide substitutions when the length-difference method of estimation is used, but the extent of underestimation is small when the site-difference method is used. (4) Evolutionary relationships of DNA sequences in finite populations. A mathematical theory on the expected evolutionary relationships among DNA sequences (nucleons) randomly chosen from the same or different populations is developed under the assumption that the evolutionary change of nucleons is determined solely by mutation and random genetic drift. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author). UMI ^

DNA-DAMAGING REACTIONS OF NEOCARZINOSTATIN (ANTIBIOTICS, DNA REPAIR, CYTOTOXICITY)

Sensitive assays utilizing a cell-free and an intracellular system were employed to study the molecular bases of the DNA-damaging reactions of neocarzinostatin (NCS). In the cell-free DNA system, super-helical form I DNA from the bacteriophage PM2 was used as the substrate. The three forms of DNA present after treatment with NCS were separated by agarose gel electrophoresis. When NCS-damaged DNA was assayed under neutral conditions, there was a progressive decrease in the amount of surviving form I DNA and a corresponding increase in form II (nicked, relaxed circular) DNA, but very little increase in form III (linear duplex) DNA. This indicates that NCS introduces primarily single-strand breaks. However later studies showed that there were some site-specific double-strand breaks mediated by NCS on PM2 DNA. Seven such specific sites were mapped on the PM2 genome. When the damage was assayed under nondenaturing alkaline conditions or with the apurinic/apyrimidinic endonuclease IV, there was a slightly greater decrease in the amount of surviving form I DNA compared with neutral conditions indicating the presence of some alkali-labile sites.^ NCS-mediated DNA damage and repair were examined with cultured Chinese hamster ovary (CHO) cells using either alkaline elution for analysis of single-strand breaks or neutral elution for analysis of double-strand breaks. Most of the strand breaks introduced by NCS were capable of being rejoined. However, there was a small amount of residual DNA damage remaining unrejoined at 24-hr after removal of the drug. The amount of residual DNA damage was higher in a CHO mutant cell line (EM9) having a higher sensitivity to killing by NCS than its parental strain (AA8). Other lesions, DNA-protein complexes and alkali-labile sites, were detected after NCS treatment but they constituted only a small fraction of the DNA damage.^ Based on the above information, it can be postulated that NCS introduces some very lethal DNA damage. It is likely that the lethal lesions are a subset of the total DNA lesions representing the residual DNA damage. This DNA damage may be composed of site-specific, unrejoinable double-strand breaks and are thus the primary lesion leading to NCS-mediated lethality.^

STUDIES OF THE MOLECULAR MECHANISMS OF CHROMOSOME ABERRATION FORMATION (DNA REPAIR, CROSSLINKS, MITOMYCIN C)

The objective of this research has been to study the molecular basis for chromosome aberration formation. Predicated on a variety of data, Mitomycin C (MMC)-induced DNA damage has been postulated to cause the formation of chromatid breaks (and gaps) by preventing the replication of regions of the genome prior to mitosis. The basic protocol for these experiments involved treating synchronized Hela cells in G(,1)-phase with a 1 (mu)g/ml dose of MMC for one hour. After removing the drug, cells were then allowed to progress to mitosis and were harvested for analysis by selective detachment. Utilizing the alkaline elution assay for DNA damage, evidence was obtained to support the conclusion that Hela cells can progress through S-phase into mitosis with intact DNA-DNA interstrand crosslinks. A higher level of crosslinking was observed in those cells remaining in interphase compared to those able to reach mitosis at the time of analysis. Dual radioisotope labeling experiments revealed that, at this dose, these crosslinks were associated to the same extent with both parental and newly replicated DNA. This finding was shown not to be the result of a two-step crosslink formation mechanism in which crosslink levels increase with time after drug treatment. It was also shown not to be an artefact of the double-labeling protocol. Using neutral CsCl density gradient ultracentrifugation of mitotic cells containing BrdU-labeled newly replicated DNA, control cells exhibited one major peak at a heavy/light density. However, MMC-treated cells had this same major peak at the heavy/light density, in addition to another minor peak at a density characteristic for light/light DNA. This was interpreted as indicating either: (1) that some parental DNA had not been replicated in the MMC treated sample or; (2) that a recombination repair mechanism was operational. To distinguish between these two possibilities, flow cytometric DNA fluorescence (i.e., DNA content) measurements of MMC-treated and control cells were made. These studies revealed that the mitotic cells that had been treated with MMC while in G(,1)-phase displayed a 10-20% lower DNA content than untreated control cells when measured under conditions that neutralize chromosome condensation effects (i.e., hypotonic treatment). These measurements were made under conditions in which the binding of the drug, MMC, was shown not to interfere with the stoichiometry of the ethidium bromide-mithramycin stain. At the chromosome level, differential staining techniques were used in an attempt to visualize unreplicated regions of the genome, but staining indicative of large unreplicated regions was not observed. These results are best explained by a recombinogenic mechanism. A model consistent with these results has been proposed.^