Passage number of cancer cell lines: Importance,intricacies, and way-forward

Cancer cell lines play a crucial role as invaluable models in cancer research, facilitating the examination of cancer progression as well as the advancement of diagnostics and treatments. While they may not perfectly replicate the original tumor, they generally exhibit similar characteristics. Low-passage cancer cell lines are generally preferred due to their closer resemblance to the original tumor, as long-term culturing can alter the genetic and molecular profiles of a cell line thereby highlighting the importance of monitoring the passage number (PN). Variations in proliferation, migration, gene expression, and drug sensitivity can be linked to PN differences. PN can also influence DNA methylation levels, metabolic profiles, and the expression of genes/or proteins in cancer cell lines. When conducting research on cancer cell lines, it is crucial for researchers to carefully select the appropriate PN to maintain consistency and reliability of results. Moreover, to ensure dependability and replicability, scientists ought to actively track the growth, migration, and gene/or protein profiles of cancer cell lines at specific PNs. This approach enables the identification of the most suitable range of PNs for experiments, guaranteeing consistent and precise results. Additionally, such efforts serve to minimize disparities and uphold the integrity of research. In this review, we have laid out recommendations for laboratories to overcome these PN discrepancies when working with cancer cell lines.     

MONOCLONAL ANTIBODIES AS MOLECULAR MARKERS FOR THE INTRACELLULAR AND CELL WALL DISTRIBUTION OF CARRAGEENAN EPITOPES IN KAPPAPHYCUS (RHODOPHYTA) DURING TISSUE DEVELOPMENT1

Carrageenan, the major cell wall carbohydrate of certain red algae, is variable in structure and gelling properties. Sequence types include gelling (kappa and iota) and nongelling (lambda) types in addition to precursors, often in hybrid molecules containing more than one precursor and/or sequence type. Molecular markers to subunits were needed to study carrageenan synthesis, cell wall organization, and the relationship between structure and function. Monoclonal antibodies were produced to carrageenan, and their specificities were determined by competitive enzyme immunoassay. Antibodies were identified with specificities related to kappa, iota, and lambda carrageenan. The patterns of immunofluorescence localization on Kappaphycus alvarezii = Eucheuma alvarezii var. tambalang (Doty) sections were distinctive for each antibody. The antibody to a kappa-related epitope labeled mature tissue strongly; antibodies to an iota-related epitope and a lambda-related epitope labeled weakly, consistent with the kappa-enriched carrageenan produced by this alga. Kappa-related epitopes were distributed throughout the wall and matrix, whereas iota-related epitopes were concentrated in the middle lamella. Lambda-related epitopes were localized primarily at the plant cuticle where kappa and iota antigens were lacking. An antibody appeared to be specific for a precursor of the gelling subunits because it showed maximal wall and intracellular labeling at the youngest developmental stage. All antibodies labeled intracellular inclusions in the transition zone between the epidermis and medulla during the development of medullary cells from the peripheral meristem in young branches. The results demonstrate the intracellular synthesis of epitopes related to all major carrageenan subunits and their differential extracellular distribution.     

ENDOSPERM DEVELOPMENT IN THE DATE PALM (PHOENIX DACTYLIFERA) (ARECACEAE)

Date seeds were sampled at regular intervals from pollination (March) to mature fruit (September) and processed for light microscopy and SDS-PAGE. Seed fresh weight rose until early June and then declined slightly through September due to a continuous decrease in water content. Cell wall formation started in May in the free nuclear endosperm and proceeded centripetally from the inner integument to the seed center. Wall thickening in each cell started in cell corners and showed a layered appearance with calcofluor white staining. It started in early June in the center of the seed and proceeded centrifugally such that the outer cells showed cell wall thickening in late June. Thickened cell walls were soft and PAS positive at inception, but staining disappeared and hardness increased during wall maturation. Cell elongation in the radial direction accompanied wall thickening. Protein body formation started after cell wall thickening and followed the same centrifugal developmental pattern. Mature protein bodies occurred in even the outermost cells by early July. No further structural changes occurred after this time. The high molecular weight storage proteins appeared in late June, which is when protein bodies had formed in all but the outer endosperm cells; however, these proteins did not appear simultaneously and minor changes in protein bands continued until maturation. α-Galactosidase activity was present in the developing endosperm and peaked at 13 wk after pollination. The data suggest that the thickened wall is deposited as a highly substituted galactomannan, but that most of the galactose side branches are clipped off presumably by α-galactosidase during cell wall polymerization.     

Citric acid fermentation by the mutant strain of theAspergillus niger resistant to manganese ions inhibition

Summary A new mutant strain,Aspergillus niger GS-III, showing resistance to manganese ions inhibition of citric acid fermentation on a sugarcane molasses containing medium was induced fromAspergillus niger KCU 520, a high citric acid-yielding strain. In submerged, surface or continuous cultures in the presence of manganese ions concentration upto 1.5 ppm the mutant strain yielded citric acid about 90 KgM^–3 . The citric acid yield was comparable to that obtained with the parental strain KCU 520 in the absence of manganese ions, but it was atleast 3-fold higher than that obtained by the latter in the presence of manganese ions. The mutant strain immobilized in calcium alginate beads was used in combination with surface-stabilized cultures for about 36-days in a continuous flow horizontal fermenter without any apparent loss in citric acid productivity. These results indicate that the manganese-resistant mutant is stable and may be used in the presence of sufficient manganese ions concentration (1.5 ppm) in the fermentation medium. This capability of the mutant strainA. niger GS-III has been correlated with greatly reduced levels (about one-thirds) of the NADP⁺ -isocitric dehydrogenase, one of the control points for citric acid accumulation.     

Interaction of heavy metal toxicants with brain constitutive nitric oxide synthase

This study was designed to evaluate thein vitro effects of transition heavy metal cations on activity of constitutive isoform of nitric oxide synthase (cNOS) in rat brain. NOS activity was determined in the cytosolic fractions of rat cerebral hemispheres by conversion of³H-L-arginine to³H-L-citrulline. Different concentrations of mercury (Hg²⁺), nickel (Ni²⁺), manganese (Mn²⁺), zinc (Zn²⁺), cadmium (Cd²⁺), lead (Pb²⁺) and calcium (Ca²⁺) were tested on NOS activity. While all the cations caused inhibition, there were differences in the apparent inhibition constants (Ki) among the cations. With the exception of calcium ion no other cation required preincubation with the enzyme preparation. These results indicate that while calcium ion modulate cNOS activity at regulatory site(s), inhibitory influence of toxic heavy metal cations may be exerted on the catalytic site(s) either by direct binding to it or by interfering with the electron transfer during catalysis.     

Addition of lutein, lycopene, or β-carotene to LDL or serum in vitro: Effects on carotenoid distribution, LDL composition, and LDL oxidation

Carotenoids are dietary antioxidants transported with plasma lipoproteins, primarily low-density lipoprotein (LDL). In this study in vitro methods were used to increase the amounts of specific, individual carotenoids in LDL. By addition of carotenoid to isolated LDL or to serum, followed by (re)isolation of the lipoproteins, samples of LDL were enriched 4- to 150-fold with lutein, 2- to 15-fold with lycopene, or 3- to 25-fold with β-carotene. Enrichment with specific carotenoids was achieved without affecting the electrophoretic mobility of the lipoprotein, its cholesterol to protein ratio, or the levels of other cartenoids or -tocopherol. The distributions among lipoproteins of carotenoid added to serum were similar, but not identical, to the distributions of the endogenous carotenoids. In particular, for added lutein, a greater proportion was found in HDL, and for added β-carotene, more was found in very low-density lipoprotein (VLDL). We then studied the effect of enriching LDL with specific carotenoids on its susceptibility to oxidation by copper ions. Lutein, β-cryptoxanthin, lycopene, and β-carotene, the four major plasma carotenoids, and -tocopherol were destroyed before the formation of lipid peroxidation products. The rates of destruction of the individual carotenoids differed; lycopene was destroyed most rapidly and lutein most slowly. Upon oxidation of β-carotene-enriched LDL, the rates of destruction of β-carotene, lycopene, and lutein were slowed and the lag times before the initiation of lipid peroxidation increased from 19 to 65 min. Neither effect was observed in LDL enriched with lutein or lycopene. Thus, β-carotene was unique among the carotenoids studied in having a small, but significant effect on LDL oxidation in vitro.     

Alterations in Phosphatidylcholine Metabolism of Stretch-Injured Cultured Rat Astrocytes

Abstract: The primary objective of this study was to determine the influence of stretch-induced cell injury on the metabolism of cellular phosphatidylcholine (PC). Neonatal rat astrocytes were grown to confluency in Silastic-bottomed tissue culture wells in medium that was usually supplemented with 10 µM unlabeled arachidonate. Cell injury was produced by stretching (5–10 mm) the Silastic membrane with a 50-ms pulse of compressed air. Stretch-induced cell injury increased the incorporation of [³H]choline into PC in an incubation time- and stretch magnitude-dependent manner. PC biosynthesis was increased three- to fourfold between 1.5 and 4.5 h after injury and returned to control levels by 24 h postinjury. Stretch-induced cell injury also increased the activity of several enzymes involved in the hydrolysis [phospholipase A₂ (EC 3.1.1.4) and C (PLC; EC 3.1.4.3)] and biosynthesis [phosphocholine cytidylyltransferase (PCT; EC 2.7.7.15)] of PC. Stretch-induced increases in PC biosynthesis and PCT activity correlated well (r = 0.983) and were significantly reduced by pretrating (1 h) the cells with an iron chelator (deferoxamine) or scavengers of reactive oxygen species such as superoxide dismutase and catalase. The stretch-dependent increase in PC biosynthesis was also reduced by antioxidants (vitamin E, vitamin E succinate, vitamin E phosphate, melatonin, and n-acetylcysteine). Arachidonate-enriched cells were more susceptible to stretch-induced injury because lactate dehydrogenase release and PC biosynthesis were significantly less in non-arachidonate-enriched cells. In summary, the data suggest that stretch-induced cell injury is (a) a result of an increase in the cellular level of hydroxyl radicals produced by an iron-catalyzed Haber-Weiss reaction, (b) due in part to the interaction of oxyradicals with the polyunsaturated fatty acids of cellular phospholipids such as PC, and (c) reversible as long as the cell's membrane repair functions (PC hydrolysis and biosynthesis) are sufficient to repair injured membranes. These results suggest that stretch-induced cell injury in vitro may mimic in part experimental traumatic brain injury in vivo because alterations in cellular PC biosynthesis and PLC activity are similar in both models. Therefore, this in vitro model of stretch-induced injury may supplement or be a reasonable alternative to some in vivo models of brain injury for determining the mechanisms by which traumatic cell injury results in cell dysfunction.     

Collagenase of a new streptomycete: Its induction and purification

Rifampin and chloramphenicol inhibited the synthesis of collagenase of Streptomyces sp. A₈, suggesting de novo synthesis. The collagenase was induced by insoluble collagen, its macromolecular fragments, gelatin, peptone, hide powder and yeast extract. Growth as well as collagenase synthesis were dependent on substrate availability. Purification of collagenase by DEAE-cellulose chromatography resulted in approximately 25-fold increase in activity (268.6 μmol glycine equivalents min^?1 mg^?1 protein) relative to the activity of the culture filtrate (10.5 μmol glycine equivalents min^?1 mg^?1 protein).