Strengths and Weaknesses of Cell Synchronization Protocols Based on Inhibition of DNA Synthesis

Synchronous cell populations are commonly used for the analysis of various aspects of cellular metabolism at specific stages of the cell cycle. Cell synchronization at a chosen cell cycle stage is most frequently achieved by inhibition of specific metabolic pathway(s). In this respect, various protocols have been developed to synchronize cells in particular cell cycle stages. In this review, we provide an overview of the protocols for cell synchronization of mammalian cells based on the inhibition of synthesis of DNA building blocks—deoxynucleotides and/or inhibition of DNA synthesis. The mechanism of action, examples of their use, and advantages and disadvantages are described with the aim of providing a guide for the selection of suitable protocol for different studied situations.

REverSe TRanscrIptase Chain Termination (RESTRICT) for Selective Measurement of Nucleotide Analogs Used in HIV Care and Prevention

Sufficient drug concentrations are required for efficacy of antiretroviral drugs used in human immunodeficiency virus (HIV) care and prevention. Measurement of nucleotide analogs, included in most HIV medication regimens, enables monitoring of short- and long-term adherence and the risk of treatment failure. The REverSe TRanscrIptase Chain Termination (RESTRICT) assay rapidly infers the concentration of intracellular nucleotide analogs based on the inhibition of DNA synthesis by HIV reverse transcriptase (RT) enzyme. Here, we introduce a probabilistic predictive model for RESTRICT and demonstrate selective measurement of multiple nucleotide analogs using DNA templates designed according to the chemical structure of each drug. We measure clinically relevant concentrations of tenofovir diphosphate (TFV-DP), emtricitabine triphosphate (FTC-TP), and azidothymidine triphosphate (AZT-TP) with agreement between experiment and theory. RESTRICT represents a new class of activity-based assays for therapeutic drug monitoring and precision dosing in HIV care and could be extended to other diseases treated with nucleotide analogs.

Effect of Cladribine on Neuronal Apoptosis: New Insight of In Vitro Study in Multiple Sclerosis Therapy

Background: Cladribine (2-CdA) can cross the blood–brain barrier, resulting in inhibition of DNA synthesis and repair and disruption of cellular proliferation in actively dividing lymphocytes. No data on effect on neurons are available. Aim: To study “in vitro” 2-CdA apoptotic effects on neurons in healthy donor and multiple sclerosis patient lymphocytes. Methods: Neuroblastoma cells were co-cultured with lymphocytes, with and without 2-CdA. Results: Apoptosis increased in lymphocytes with 2-CdA; increase was also observed when lymphocytes were cultured with neuronal cells. However, neurons were not affected by 2-CdA for apoptosis. Conclusions: 2-CdA causes peripheral and central lymphocyte death preserving neurons, with a reasonable impact on inflammation and neuroprotection.

Combination Therapy in Melasma with Lentigo Solaris

Background: Melasma and lentigo solaris are common, recurrent, and refractory acquired hyperpigmentation disorder. In spite of variety of therapeutic options available for this cosmetically disfiguring condition, the treatment of this condition remains a challenge. Azelaic acid (AA) is a depigmenting agent which acts by inhibition of DNA synthesis and mitochondrial enzymes, thereby inducing direct cytotoxic effects on melanocytes. Glycolic acid (GA) peel is one of the most versatile agents in the treatment of melasma and lentigo solaris. GA peels alone or in combination with topical hypopigmenting agents has shown encouraging results. However, there is paucity of controlled trial demonstrating the efficacy of GA peels in conjunction with topical AA. Case: A 42-years-old female, works as a street vendor, came with dark brown spots on both cheeks, nose, chin and forehead that spreads to whole face since one year ago. She had a history of using contraceptives. From the dermatological examination, there were multiple well-circumscribed, irregular hyperpigmented macules that asymmetrical, with size ranging from lenticular to plaque on the maxillary, left buccalis, mentalis and frontalis region. We also found a numular dark brown hyperpigmented macules on right zygoma. She was diagnosed with melasma and lentigo solaris. The Melasma Area Severity Index (MASI) score was 25.6, which classified as moderate melasma. She was treated with 20% azaleic acid cream twice a day, broadspectrum sunscreen with SPF 50 and GA 20% peeling. Result: After 6 weeks of treatment, there were significant improvement in both melasma and lentigo solaris.

Folate rescues vitamin B12 depletion-induced inhibition of nuclear thymidylate biosynthesis and genome instability

Clinical vitamin B12 deficiency can result in megaloblastic anemia, which results from the inhibition of DNA synthesis by trapping folate cofactors in the form of 5-methyltetrahydrofolate (5-methylTHF) and subsequent inhibition of de novo thymidylate (dTMP) biosynthesis. In the cytosol, vitamin B12 functions in the remethylation of homocysteine to methionine, which regenerates THF from 5-methylTHF. In the nucleus, THF is required for de novo dTMP biosynthesis, but it is not understood how 5-methylTHF accumulation in the cytosol impairs nuclear dTMP biosynthesis. The impact of vitamin B12 depletion on nuclear de novo dTMP biosynthesis was investigated in methionine synthase-null human fibroblast and nitrous oxide-treated HeLa cell models. The nucleus was the most sensitive cellular compartment to 5-methylTHF accumulation, with levels increasing greater than fourfold. Vitamin B12 depletion decreased de novo dTMP biosynthesis capacity by 5–35%, whereas de novo purine synthesis, which occurs in the cytosol, was not affected. Phosphorylated histone H2AX (γH2AX), a marker of DNA double-strand breaks, was increased in vitamin B12 depletion, and this effect was exacerbated by folate depletion. These studies also revealed that 5-formylTHF, a slow, tight-binding inhibitor of serine hydroxymethyltransferase (SHMT), was enriched in nuclei, accounting for 35% of folate cofactors, explaining previous observations that nuclear SHMT is not a robust source of one-carbons for de novo dTMP biosynthesis. These findings indicate that a nuclear 5-methylTHF trap occurs in vitamin B12 depletion, which suppresses de novo dTMP biosynthesis and causes DNA damage, accounting for the pathophysiology of megaloblastic anemia observed in vitamin B12 and folate deficiency.

9-(2′-Deoxy-2′-Fluoro-β-d-Arabinofuranosyl) Adenine Is a Potent Antitrypanosomal Adenosine Analogue That Circumvents Transport-Related Drug Resistance

ABSTRACT Current chemotherapy against African sleeping sickness, a disease caused by the protozoan parasite Trypanosoma brucei, is limited by toxicity, inefficacy, and drug resistance. Nucleoside analogues have been successfully used to cure T. brucei-infected mice, but they have the limitation of mainly being taken up by the P2 nucleoside transporter, which, when mutated, is a common cause of multidrug resistance in T. brucei. We report here that adenine arabinoside (Ara-A) and the newly tested drug 9-(2′-deoxy-2′-fluoro-β-d-arabinofuranosyl) adenine (FANA-A) are instead taken up by the P1 nucleoside transporter, which is not associated with drug resistance. Like Ara-A, FANA-A was found to be resistant to cleavage by methylthioadenosine phosphorylase, an enzyme that protects T. brucei against the antitrypanosomal effects of deoxyadenosine. Another important factor behind the selectivity of nucleoside analogues is how well they are phosphorylated within the cell. We found that the T. brucei adenosine kinase had a higher catalytic efficiency with FANA-A than the mammalian enzyme, and T. brucei cells treated with FANA-A accumulated high levels of FANA-A triphosphate, which even surpassed the level of ATP and led to cell cycle arrest, inhibition of DNA synthesis, and the accumulation of DNA breaks. FANA-A inhibited nucleic acid biosynthesis and parasite proliferation with 50% effective concentrations (EC50s) in the low nanomolar range, whereas mammalian cell proliferation was inhibited in the micromolar range. Both Ara-A and FANA-A, in combination with deoxycoformycin, cured T. brucei-infected mice, but FANA-A did so at a dose 100 times lower than that of Ara-A.

Effects of water soluble oncostatic fraction from Rheum officinale Baill. rhizomes on Allium cepa root meristem. I. The mitotic activity

The effects of the oncostatic extracts from <i>Rheum officinale</i> rhizomes on the activity of meristematic cells from <i>Allium cepa</i> roots were investigated. A statistically significant decrease of the IM value was noted as well as of the total number of mitoses during incubation. The disturbances in the course of mitosis and cytokinesis are described and discussed. The kind of disturbances during postincubation points to damage of the S and G2 phases of the interphase nuclei. Cytochemical and autoradiographic studies demonstrated a diminished intensity of staining of DNA and RNA and inhibition of DNA synthesis during incubation, this leading in tum to a lower intensity of protein staining in postincubation. Disturbances in mitosis and cytokinesis after treatment wth 2,6-dihydroxyantraquinone, supposed to be the antimitotically active compound of the extract, are the same as those produced by the total water soluble fraction.

The effect of the edein complex on the activity of apical root meristems of Allium cepa L.

The effect of a mixture of edeins in water solution on the meristematic activity of <em>Allium cepa</em> L. root tips was studied during the incubation and postincubation periods. Reduction of the elongation growth of the root tips and a strong decrease in. the number of mitoses was observed what, as cytochemical and autoradiographical studies have shown, is due to the inhibition of DNA synthesis. DNA synthesis is completely inhibited after 24 hrs of incubation. Changes caused by 25 ppm concentration of the solution are reversible during postincubation. The concentration of 50 ppm produces irreversible damage. The main direction of cytochemical changes caused by the action of edeins on the meristematic cells is in good accordance with those recorded for bacterial cells (Kuryło-Borowska 1962, Borowski and Chmara 1968).