Paul A. Kitos

Estimating the number of viable animal cells in multi-well cultures based on their lactate dehydrogenase activities

A method is described for estimating the numbers ofanimal cells in multi-well culture by simultaneouslymeasuring the lactate dehydrogenase activity of thetotal culture and the medium. The difference betweenthe two reflects the dehydrogenase content of thecells and correlates with cell number. This LDH/INTmethod was tested using several lines of normal andtransformed suspension and adherent cells. Thelactate dehydrogenase activities of duplicate cultureswere determined colourimetrically using reactioncocktails containing lactate, NAD+, diaphorase,and p-iodonitrotetrazolium violet, with and withoutTriton X-100. The difference in absorbance at 490 nm(ΔA490 = A490, test – A490, control) was used to calculate the lactatedehydrogenase activity of the total culture (+ Triton)and the medium (– Triton). The cellular lactatedehydrogenase activity (difference between totaland medium dehydrogenaseactivities) was proportional to viable cell number. The effects on cell growth of four metabolicinhibitors, sodium azide, actinomycin D,cycloheximide, and taxol, were determined using theLDH/INT assay and direct cell counting. The inhibitorconcentrations that caused decreases in the LDHactivity and cell number by 50% were similar. TheLDH/INT assay is quick and sensitive, works equallywell for adherent and suspension cells, and providesinformation about LDH activities of both the mediumand cells. It is particularly useful for screeningpotential cell-growth inhibitors.

A demonstration of the intrinsic importance of stabilizing hydrophobic binding and non-convalent van der waals contacts dominant in the non-covalent CC-1065/B-DNA binding

The comparative DNA binding properties and cytotoxic activity of CDPIn methyl esters (n = 1-5) vs. PDE-In methyl esters (n = 1-3) are detailed in studies which provide experimental evidence for the intrinsic importance of stabilizing hydrophobic binding and non-covalent van der Waals contacts dominant in the CC-1065/B-DNA minor groove binding. High affinity minor groove binding to DNA was established through: (1) the observation of CDPI3 binding (UV) but not unwinding of supercoiled DNA (phi 174 RFI DNA) thus excluding intercalative binding; (2) the observation of CDPI3 binding to T4 phage DNA (UV, delta Tm) in which the major groove is occluded by glycosylation thus excluding major groove binding; (3) the observation of salt (Na+) concentration independent high affinity CDPI3 binding to poly(dA . poly(dT) thus excluding simple electrostatic binding to the DNA phosphate backbone; and further inferred through (4) the observation of an intense induced dichroism (ICD, poly(dA) . poly(dT) and poly(dG) . poly(dC) [phi]23(358) = 24,000 and 23,500). This high affinity minor groove binding is sufficient to produce a potent cytotoxic effect.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA alkylation properties of the duocarmycins: (+)-duocarmycin A, epi-(+)-duocarmycin A, ent-(−)-duocarmycin A and epi,ent-(−)-duocarmycin A

Abstract A study of the comparative in vitro cytotoxic activity and DNA alkylation properties of both enantiomers of the two diastereomers of (+)-duocarmycin A are detailed. The DNA alkylation efficiency and in vitro cytotoxic potency of the natural enantiomers ((+)-duocarmycin A epi -(+)-duocarmycin A, 3-8x) exceed those of the unnatural enantiomers ( ent -(−)-duocarmycin A, epi,ent -(−)-duocarmycin A) by at least 100x.

Synthesis and preliminary evaluation of (+)-CBI-indole2: an enhanced functional analog of (+)-CC-1065

Abstract The synthesis and comparative preliminary evaluation of (+)-CBI-indole 2 ( 3 ) are detailed in efforts that further demonstrate a direct relationship between the chemical stability of the agents and their biological potency/DNA alkylation intensity.

Teratogenic effects of cholinergic insecticides in chick embryos. I. Diazinon treatment on acetylcholinesterase and choline acetyltransferase activities.

Abstract Teratogenic effects of diazinon were assessed morphologically and correlated with values determined by radiometric determinations for acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities. Diazinon, at doses ranging from 23 to 1854 μg/chick egg, was injected on Day 3 of incubation, and enzyme activities in hindlimb, wing, and brain were measured on Days 6–20 of incubation. Generally, these organs displayed similar patterns of enzyme alteration. With an injected dose of 200 μg diazinon per egg, AChE activity was inhibited about 90% at Days 6–8, about 50% at Day 10, and not at all at Day 13. On the other hand, ChAT activity was not significantly different between diazinon- and corn oil-injected embryos. The threshold dose for type II teratogenic signs (such as wry neck and short neck) was higher than for type I signs (such as micromelia and abnormal feathering). Morphological studies, using atropine and gallamine, suggested that nicotinic but not muscarinic receptors may be involved in the mechanism of diazinon-induced type II malformations. Nicotinamide, which prevented type I malformations, did not prevent the diazinon-induced AChE inhibition. From the above findings, we confirm and extend the finding of others, that the cholinergic dysfunction does not temporally correlate with the type I teratogenic effects.

Teratogenic effects of cholinergic insecticides in chick embryos--II. Effects on the NAD content of early embryos.

Abstract Chick embryos at 72 hr of incubation were exposed in ovo (0.2 or 1.0 mg per egg) to the organophosphate insecticide diazinon (DZN) or dicrotophos (DCP) and examined at days 5, 7 and 10 for visible defects and changes in the protein and NAD content of the whole organism, their wings and legs. Skeletal changes of the spinal column and legs were scarcely detectable on day 5 but were readily apparent by day 10. the NAD contents of the embryos and their limbs were normal up to day 5, slightiy reduced by day 7, and greatly reduced by day 10, while the wet weights and protein contents were virtually normal over this time span. Nicotinamide (8 μmoles egg on day 3) increased the NAD content of the embryos by 20–30 per cent. If administered along with DZN or DCP, it produced an even greater increase by day 10 and minimized the insecticide-induced leg deformities. A reactivator of organophosphate-inhibited acetylcholine esterase, 2-pyridinealdoxime methochloride (2-PAM), did not affect the NAD content of control embryos during these early times. If given with the insecticide, however, it prevented the expected decrease in NAD level. Some of the effects of the cholinergic insecticides and their reversing agents on the NAD content of young chick embryos are considered in terms of their possible mechanisms of action and teratogenic implications.

10-formyl-5,8,10-trideazafolic acid (10-formyl-TDAF): A potent inhibitor of glycinamide ribonucleotide transformylase

The synthesis of 10-formyl-5,8,10-trideazafolic acid (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) is reported. The target compound was prepared by a convergent synthesis utilizing the alkylation of hydrazone 5 with benzylic bromide 6 to construct the core heterocycle 7. The aldehyde 3 and related agents were evaluated as inhibitors of purN GAR Tfase and avian AICAR Tfase. Compound 3 exhibited potent inhibition of GAR Tfase with a Ki of 0.26 +/- 0.05 microM. In contrast, 3 exhibited more moderate inhibition of aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase), with Ki of 7.6 +/- 1.5 microM.

A potent, simple derivative of an analog of the CC-1065 alkylation subunit

Abstract A simple semicarbazide derivative of CBI, an analog of the authentic CPI alkylation subunit of CC-1065, exhibits enhanced in vitro cytotoxic potency and DNA alkylation properties ( ca. 100x) derived from noncovalent electrostatic DNA binding.

Design, synthesis and evaluation of bouvardin, deoxybouvardin and RA-I-XIV pharmacophore analogs

The synthesis and in vitro cytotoxic evaluation of a key set of cycloisodityrosine subunit analogs of deoxybouvardin and RA-VII are detailed and constitute a complete investigation of the natural product pharmacophore. The studies illustrate that the 18-membered ring tetrapeptide potentiation of the cytotoxic activity of cycloisodityrosine is not likely to be due to simple alteration or constraint of the conformation of the 14-membered cycloisodityrosine subunit and that simple derivatization of cycloisodityrosine may not provide the same potentiation.

Regulation of glutamine synthetase in L cells by cortisol andl-glutamine

SummaryThe glutamine synthetase system of strain L mouse cells is regulated by at least two natural substances: glutamine and a glucocorticoid hormone. In a chemically defined cell culture system, cortisol or dexamethasone promotes intracellular accumulation of the synthetase by a process which depends upon genetic transcription. The accumulation is most rapid if the hormone is added to a glutamine deficient culture medium. High concentrations of glutamine promote rapid disappearance of the enzyme by a mechanism which depends on unimpaired protein synthesis. Ways in which the two regulatory substances collaborate to influence the synthetase level and other growth-related processes of L cells in culture are discussed.