Interaction of two second generation platinum antitumor drugs with mouse thymocytes.

The effects of two second generation platinum drugs, cis-diammine-1, 1-cyclobutane dicarboxylate platinum(II) and cis-dichloro-trans-dihydroxybis(isopropylammine)platinum(IV) , were studied on thymocyte nucleosomes, calf thymus DNA, and intact murine thymocytes. In contrast to cis-dichlorodiamineplatinum(II) (cis-DDP), the binding of cis-diammine-1,1-cyclobutarol dicarboxylate platinum(II) or cis-dichloro-trans-dihydroxybis(isopropylammine)platinum(IV) to nucleosomes or DNA was markedly diminished at commonly used pharmacological doses. Since comparable amounts of the drugs were bound to whole thymocytes, we investigated possible membrane sites of action. The fluorescent probes diphenylhexatriene and trimethylammoniumdi-phenylhexatriene were used to measure membrane fluidity. In the whole cell, marked decreases in anisotropy constants of 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene-p-toluene sulfonate were observed after treatment with cis-diammine-1,1-cyclobutarol dicarboxylate platinum(II) and cis-dichloro-trans-dihydroxybis(isopropylammine)platinum(IV) , but not cis-DDP, at 37 degrees C at therapeutic drug concentrations. This effect was observed only in intact thymocytes, not in isolated plasma membranes or liposomes treated similarly with the drugs. The fluorescent properties of the phospholipid probe, 1-acyl-2-(N-4-nitrobenzo-2-oxa, 1,3-diazole)aminocaproylphosphatidylcholine, were altered only by cis-DDP, whereas those of 1-acyl-2-(6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl ) phosphatidylethanolamine were altered by all three drugs. The increase in the fluorescence intensity per cell after reaction with the drugs was measured with the flow cytometer. The results suggest that alterations in membrane fluidity are produced by the hydrophobic second generation drugs, whereas changes observed via the probe based on phosphatidylcholine were produced only with cis-DDP. Since cis-DDP has a known avidity for proteins, the observed effects may be related to an altered protein-phospholipid (phosphatidylcholine) interaction. The changes in membrane structure described here may relate directly to cytotoxicity or may effect changes enabling the drug to enter the cell to find its intracellular targets.

Effects of adriamycin on supercoiled DNA and calf thymus nucleosomes studied with fluorescent probes.

The interaction of the antitumor drug Adriamycin with nucleotides, polynucleotides, RNA, calf thymus nucleosomes, and DNA (including pBR322 supercoiled DNA) has been studied using fluorescent probes. The lanthanide terbium is known to interact with guanine and xanthosine to produce high fluorescence enhancement. The nature of the interaction of the lanthanide with the heterocyclic ring in guanine appears to involve both the C-2 and N-7 groups. A striking decrease in fluorescence enhancement was observed with all of the polynucleotides, RNA, DNA, and nucleosomes after treatment with Adriamycin at molar ratios of 1:200 or less. It appears that Adriamycin interacts with the guanine ring, displacing or preventing terbium access to its second site of binding. However, with supercoiled DNA and nucleosomes, the displacement followed a destabilization of the helix at very low drug concentrations. The binding affinities of calf thymus DNA, pBR322 DNA, and calf thymus nucleosomes at 37 degrees for Adriamycin were of the same order of magnitude. Reaction with N-pyrene maleimide, a fluorescent probe which binds to histone H3, showed that Adriamycin interacted with the nucleosome to increase the binding of the probe (only, however, at drug ratios far greater than those required to produce effects with DNA). No compositional changes of supercoiled or nucleosomal DNA or nucleosomal histones were observed by agarose gel or sodium dodecyl sulfate:polyacrylamide gel electrophoresis, respectively. The classic intercalating agent, ethidium bromide, produced minimal displacement of the lanthanide from DNA, although an effect with RNA at high drug concentrations was observed.

Interaction of three second-generation anthracyclines with polynucleotides, RNA, DNA, and nucleosomes.

The interaction of three second-generation anthracycline derivatives with polynucleotides, supercoiled DNA, and calf thymus nucleosomes has been studied by terbium fluorescence measurements and agarose gel electrophoresis. It was shown that, as expected, N-trifluoroacetyladriamycin-14-valerate had little detectable effect on the fluorescence of this guanine-specific probe except for polydisperse calf thymus linear DNA fragments. The soluble analogue, N-trifluoroacetyladriamycin-14-O-hemiadipate, did show marked effects on terbium fluorescence with all nucleic acids and nucleosomes, but the effects were generally not as striking as were those observed with the epimer, 4'-epi-Adriamycin, which tended to produce a similar effect to its parent drug, Adriamycin, showing that a marked change in the hexose ring did not appreciably affect the interaction of the drug with DNA. Changes in the electrophoretic mobility of supercoiled pBR322 DNA were observed only at very high drug concentrations, much higher than those required with Adriamycin or actinomycin D. The effect was a smearing of the form I DNA and the production of some circular relaxed form II DNA. The drugs produced the effect in the following order: Adriamycin greater than N-trifluoroacetyladriamycin-14-O-hemiadipate greater than or equal to epi-Adriamycin. N-Trifluoroacetyladriamycin-14-valerate had little effect, even at very high drug concentrations (1:2, drug:DNA ratios).

The binding of actinomycin D and Adriamycin to supercoiled DNA, single-stranded DNA and polynucleotides.

The effect of actinomycin D and adriamycin on synthetic polynucleotides, single-stranded viral DNA and supercoiled DNA has been studied employing the fluorescent probe, terbium. Marked displacement of the probe was observed when any deoxyribose-containing polynucleotide was pretreated with either drug. With supercoiled DNA, an unwinding of the supercoil was observed at very low drug concentrations (at approx. 1:500 molar ratio of drug:DNA) prior to the displacement of the terbium. This unwinding was visualized by agarose gel electrophoresis at molar ratios of approx. 1:200. The effect was more apparent and occurred at lower drug: DNA ratios with actinomycin D than with adriamycin. Unlike cis-dichlorodiammine platinum(II), actinomycin D did not protect pBR322 DNA from cleavage at its BamHI site. The hydrolysis of phi chi 174 DNA by a series of G-C-specific restriction nucleases (including HhaI, HpaII and HaeIII) was also not affected by prior treatment of the DNA with actinomycin D.

The differential effects produced by cis- and trans-DDP on DNA in calf thymus nucleosomes.

Calf thymus nucleosomes containing H1 were treated with dichlorodiammineplatinum (DDP) at low binding ratios (r = 0.05-0.15). Change in the electrophoretic mobility of the extracted nucleosomal DNA was observed following treatment with cis-DDP and little change with trans-DDP. There was a decrease in the electrophoretic mobility of the nucleosomal DNA as well as obliteration of the nucleosomal repeat distance. The fluorescence intensity of terbium binding to the extracted DNA showed minimal change following drug treatment. However, the thermal melting behavior of the nucleosomal DNA was altered to a greater extent following cis-DDP treatment at 280 rather than 260 nm and a destabilization of the DNA helix was observed. These data suggest that in the whole nucleosome, cis-DDP produces greater structural effects on the packaged DNA than trans-DDP, although similar amounts of drug are bound with both isomers.

Human papillomaviruses and cervical cancer: analysis of histopathologic features associated with different viral types.

The histopathologic features of 41 cervical carcinomas were correlated with the presence of human papillomavirus (HPV). Southern blots of DNA extracted from the tumors were hybridized with 32P-labeled type specific probes for HPV 6, 11, 16, 18, and 31. HPV was found in 26/41 (63%) of the tumors. The HPV types were: HPV 16 in 17 tumors (41%), HPV 18 in six tumors (15%) and HPV 31 in two tumors (5%). No tumor hybridized to either HPV 6 or HPV 11. HPV was identified in all histologic subtypes of cervical carcinoma; however, different HPV types were associated with specific histologic features. HPV 18 was identified in four of eight adenocarcinomas, while HPV 16 was found in only one. HPV 16 was most strongly associated with the keratinizing tumors. It was found in 10/13 (77%) of the large cell keratinizing (LCK) and in only 4/16 (25%) of the large cell nonkeratinizing cervical carcinomas (LCNK). A mucoepidermoid with extensive keratinization and pearl formation also contained HPV 16. One of three additional adenosquamous carcinomas had HPV 31, as did one LCNK tumor. In one LCK tumor, a HPV was identified that hybridized to both HPV 16 and 18. The LCNK group contained the highest percentage of tumors in which no papillomavirus DNA was identified (9/16 lacked HPV DNA). No papillomavirus was detected in six tumors from other sites or in five cervical specimens with no histologic evidence of HPV infection. These data indicate that HPV is involved in all major histologic types of cervical carcinoma, and suggest that the different HPV types transform slightly different cell populations, or that transformation by HPV 18 tends to induce adeno-differentiation while HPV 16 leads to squamous maturation.

The differential effects produced by daunomycin and adriamycin on RNA, polynucleotides, single stranded, supercoiled DNA, and nucleosomes.

Terbium, a sensitive probe whose fluorescence is strongly enhanced when bound to unpaired guanine and xanthine bases, has been employed to study the effects of adriamycin and daunomycin on a variety of nucleotide substrates. After treatment with either drug at concentrations of less than or equal to 1:500, the fluorescence of the probe was substantially abrogated. Daunomycin, however, produced a markedly greater effect than adriamycin with rRNA, linear calf thymus DNA, and polyriboguanylic acid. The difference between the drugs was experimentally significant, suggesting that changing the C9 side group from a methyl (daunomycin) to an alcohol (adriamycin) may result in a changed base sequence specificity. The distinction was also evident when changes in electrophoretic mobility of supercoiled and nucleosomal DNA was monitored, but only at much higher (1:25) drug:DNA ratios.