Cell Proliferation In Tissue Culture Research Articles

Effect of Tryptophan and Kynurenine on Cell Proliferation in Tissue Culture of the Cerebral Cortex in Young and Old Rats

Metabolism disorders of L-kynurenine, which is an intermediate product of the decomposition of genetically encoded L-tryptophan amino acid, is a component in the development of a number of neuropathological processes. The effect of tryptophan and kynurenine on cell proliferation has been studied in the organotypic culture of cerebral cortex tissue in young and old rats. Tryptophan at an effective concentration (0.05 ng/mL) oppressed cell proliferation in the cerebral cortex in young and old rats by 35 and 18%, respectively. However, cell proliferation was stimulated under the action of kynurenine (1 ng/mL), and it was more pronounced in cerebral cortex explants in old rats (by 22% as compared with the control group). The obtained data on kynurenine properties for the stimulation of cell proliferation in the cerebral cortex in old animals is promising for the creation of new medicinal nootropic drugs in age-associated diseases of the nervous system.

CBAP modulates Akt-dependent TSC2 phosphorylation to promote Rheb-mTORC1 signaling and growth of T-cell acute lymphoblastic leukemia

High-frequency relapse remains a clinical hurdle for complete remission of T-cell acute lymphoblastic leukemia (T-ALL) patients, with heterogeneous dysregulated signaling profiles—including of Raf-MEK-ERK and Akt-mTORC1-S6K signaling pathways—recently being implicated in disease outcomes. Here we report that GM-CSF/IL-3/IL-5 receptor common β-chain-associated protein (CBAP) is highly expressed in human T-ALL cell lines and many primary tumor tissues and is required to bolster leukemia cell proliferation in tissue culture and for in vivo leukemogenesis in a xenograft mouse model. Downregulation of CBAP markedly restrains expansion of leukemia cells and alleviates disease aggravation of leukemic mice. Transcriptomic profiling and molecular biological analyses suggest that CBAP acts upstream of Ras and Rac1, and functions as a modulator of both Raf-MEK–ERK and Akt-mTORC1 signaling pathways to control leukemia cell growth. Specifically, CBAP facilitated Akt-dependent TSC2 phosphorylation in cell-based assays and in vitro analysis, decreased lysosomal localization of TSC2, and elevated Rheb-GTP loading and subsequent activation of mTORC1 signaling. Taken together, our findings reveal a novel oncogenic contribution of CBAP in T-ALL leukemic cells, in addition to its original pro-apoptotic function in cytokine-dependent cell lines and primary hematopoietic cells, by demonstrating its functional role in the regulation of Akt-TSC2-mTORC1 signaling for leukemia cell proliferation. Thus, CBAP represents a novel therapeutic target for many types of cancers and metabolic diseases linked to PI3K-Akt-mTORC1 signaling.

IRFP is a sensitive marker for cell number and tumor growth in high-throughput systems

GFP and luciferase are used extensively as markers both in vitro and in vivo although both have limitations. The utility of GFP fluorescence is restricted by high background signal and poor tissue penetrance. Luciferase throughput is limited in vitro by the requirement for cell lysis, while in vivo, luciferase readout is complicated by the need for substrate injection and the dependence on endogenous ATP. Here we show that near-infrared fluorescent protein in combination with widely available near-infrared scanners overcomes these obstacles and allows for the accurate determination of cell number in vitro and tumor growth in vivo in a high-throughput manner and at negligible per-well costs. This system represents a significant advance in tracking cell proliferation in tissue culture as well as in animals, with widespread applications in cell biology.

Effects of combinations of amino acids on cell proliferation in tissue culture of old rats

In organotypic tissue culture, we studied the effects of combinations of a standard amino acid at an effective concentration of 0.05 ng/mL on cell proliferation processes in explants of the spleen, myocardium, pancreas, and cerebral cortex from 24-month-old rats. Combinations of two amino acids, one of which stimulated proliferation while the other inhibited it, induced the proliferation of cells in tissue culture. These effects were 8–10% higher than that of stimulating the amino acid. These data may be used to develop new effective dipeptides, which can be used to improve regenerative tissue capabilities during aging.

Abstract IA10: How the p53 pathway controls the response to chemotherapy

Abstract Intensive drug discovery activity in both the commercial and academic sector has produced a number of small molecules that can potently regulate the p53 pathway in vivo and in vitro. The availability of these tools along with precise genetic models is allowing a re-examination of the role of the p53 response in Chemotherapy and Radiotherapy. Some chemotherapeutic agents such as actinomycin D have been found to act in an extraordinarily p53 specific manner, while others, such as doxorubicin, while still activating the p53 response actually kill cancer cells by largely p53 independent mechanism. Indeed the p53 response can reduce their effectiveness by inducing a p21 dependant cell cycle arrest. The temporal sequence and kinetics of drug action have a dramatic effect on outcome when p53 activating small molecules such as Nutlin are combined with chemotherapeutic drugs. We have synthesized new p53 activating molecules using peptide stapling methods and generated Zebra fish and mouse models to study the p53 response in normal tissues. These studies suggest that the p53 response is constitutively activated at a low level that is not incompatible with cell proliferation in tissue culture and in tumors in vivo but is not activated in many normal tissues even when they are rapidly proliferating. This low level activation may synergize with Mdm2 binding drugs to enhance their specific therapeutic index and offers scope for the further exploitation of tumor activated p53 stress pathways to enhance the selectivity of chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr IA10.

Low-dose naltrexone suppresses ovarian cancer and exhibits enhanced inhibition in combination with cisplatin

Ovarian cancer is the leading cause of death from gynecological malignancies. Although initial therapeutic modalities are successful, 65% of these women relapse with only palliative treatments available thereafter. Endogenous opioids repress the proliferation of human ovarian cancer cells in vitro, and do so in a receptor-mediated manner. The present study examined whether modulation of opioid systems by the opioid antagonist naltrexone (NTX), alone or in combination with standard of care therapies (taxol/paclitaxel, cisplatin), alters human ovarian cancer cell proliferation in tissue culture and tumor progression in mice. Administration of NTX for six hours every two days, but not continuously, reduced DNA synthesis and cell replication from vehicle-treated controls in tissue culture. Moreover, brief exposure to NTX in combination with taxol or cisplatin had an enhanced anticancer action. Mice with established ovarian tumors and treated with a low dosage of NTX (LDN), which invokes a short period of opioid receptor blockade, repressed tumor progression in a non-toxic fashion by reducing DNA synthesis and angiogenesis but not altering cell survival. The combination of LDN with cisplatin, but not taxol, resulted in an additive inhibitory effect on tumorigenesis with enhanced depression of DNA synthesis and angiogenesis. LDN combined with cisplatin alleviated the toxicity (e.g. weight loss) associated with cisplatin. LDN treatment upregulated the expression of the opioid growth factor (OGF, chemical term ([Met(5)]-enkephalin) and its receptor, OGFr. Previous tissue culture studies have reported that OGF is the only opioid peptide with antiproliferative activity on ovarian cancer cells, with OGF action mediated by OGFr. Thus, the common denominator of intermittent opioid receptor blockade by short-term NTX or LDN on ovarian cancer proliferation and tumorigenesis recorded herein appears to be related to the OGF-OGFr axis. These preclinical data may offer a non-toxic and efficacious pathway-related treatment that can benefit patients with ovarian cancer.

Pseudo-likelihood estimation for discretely observed multitype Bellman–Harris branching processes

This paper presents a method for parametric estimation in the class of multitype Bellman–Harris branching processes when the data consist of cell counts collected on several colonies observed at discrete time points. This sampling scheme arises frequently in biology to analyze cell proliferation in tissue culture. We investigate the use of a pseudo-likelihood approach in this context. It is defined from the mean vectors and variance–covariance matrices of the numbers of cells. The proposed estimator is strongly consistent and asymptotically normal as the number of observed colonies goes to infinity. In situations where these two moments have no closed-form expressions their values can be replaced by simulation-based approximations. The resulting simulated pseudo-maximum likelihood estimator is asymptotically equivalent to the pseudo-maximum likelihood estimator as the number of simulation increases. We illustrate the proposed method via two examples, and evaluate its finite sample properties through simulations.

Stable lower PAR expression decreased DU145 prostate cancer cell growth in SCID mice.

PAR is a novel gene ubiquitously expressed in normal and malignant tissues with a trend towards higher expression in tumor cells. PAR biological function is unknown. Here we report the effect of lowering PAR expression on in vitro and in vivo proliferation of DU145 cells. Decreased PAR expression was achieved by stable transfection of DU145 cells with antisense PAR cDNA cloned in pCMV-Script expression vector. The proliferative potential of DU145 transfectants was studied by cell counts, colony formation in soft agar, flow cytometry, and growth in severe combined immunodeficient (SCID) mice. DU145 transfectants exhibited a decreased cell proliferation in tissue culture and a low efficiency of colony formation in soft agar. Flow cytometry revealed an arrest of these cells in G2-M phase of mitotic cycle. A dramatic decrease of tumor growth was observed when DU145 transfectant cells were inoculated in SCID mice, compared with controls. Histological examination of these tumors showed a marked decrease in cell density and in number of mitoses while control tumors showed a high cell density and numerous mitoses. The data presented here provide the first evidence for PAR gene cellular function and its possible implication in malignant transformation.

HUMAN RENAL CELL CANCER PROLIFERATION IN TISSUE CULTURE IS TONICALLY INHIBITED BY OPIOID GROWTH FACTOR

Peptide growth factors alter cellular events by binding to specific receptors. One group of peptides, the endogenous opioids, is important in the growth of normal and neoplastic tissue. [Met5]enkephalin, also termed opioid growth factor (OGF), is a tonically active inhibitory factor that interacts with the OGF receptor, OGFr, formerly identified as Greek zeta (zeta) and appears to be autocrine produced by human cancer cells. This study examined the hypothesis that OGF directly inhibits proliferation of renal cell carcinoma in tissue culture. Human renal cancer cells (Caki-2) were grown using routine tissue culture techniques. A variety of natural and synthetic opioids including OGF, opioid antagonists, and opioid antibodies were added to renal cancer cell cultures to determine role of these peptides in renal cell carcinoma. The experiments were repeated in serum-free media, and with 4 other human renal cancer cell lines: Caki-2, A498, SN12C, and ACHN. Immunocytochemistry was performed to examine the presence of OGF and its receptor. OGF was the most potent opioid peptide to influence human renal cell carcinoma. OGF depressed growth within 12 hours of treatment, with cell numbers subnormal by up to 48% of control levels. OGF action was receptor mediated, reversible, not cytotoxic, neutralized by antibodies to the peptide, and detected in the human renal cell carcinoma lines examined. OGF appeared to be autocrine produced and secreted, and was constitutively expressed. Both OGF and its receptor were detected in these cells. OGF tonically inhibits renal cancer cell proliferation in tissue culture, and may play a role in the pathogenesis and management of human renal cell cancer.

Human pancreatic cancer cell proliferation in tissue culture is tonically inhibited by opioid growth factor.

Pancreatic adenocarcinoma is a fatal malignancy that ranks as the fourth most common cause of cancer-related mortality in the United States. The median survival after diagnosis is 3-6 months, with a 5-year survival rate of 3% or less. In spite of treatment efforts of surgery, radiation, and chemotherapy, the survival rate remains unchanged. In this study, we discovered that an endogenous opioid peptide, [Met5]-enkephalin, inhibited the growth of human pancreatic cancers in vitro; in view of this pentapeptide's action it has been termed opioid growth factor (OGF). OGF was found to be constitutively expressed, autocrine produced, and tonically capable of suppressing cell replication in an opioid receptor mediated manner. Growth inhibition was dose-related, reversible, not cytotoxic, and independent of serum. All 4 pancreatic cancer cell lines examined, representative of poor to well-differentiated neoplasias, exhibited growth regulation by OGF. Immunocytochemical studies detected both OGF and its related receptor, zeta, in the cytoplasm of log phase cells. Radioimmunoassays revealed that OGF was produced and secreted by the cells. These data suggest that a native opioid peptide, OGF, interacts with a novel opioid receptor, zeta, to arrest the growth of human pancreatic cancer.

Inhibition of experimental neointimal hyperplasia and thrombosis depends on the type of vascular injury and the site of drug administration.

Heparin inhibits vascular smooth muscle cell proliferation in tissue culture and limits neointimal hyperplasia after experimental arterial injury but has been ineffective in reducing clinical restenosis. We examined how this discrepancy might reflect suboptimal drug-tissue interactions and/or differences in the vascular response to injury. Intravenous infusion was compared with local administration of heparin to injured rabbit iliac arteries either from drug-impregnated polymeric controlled release matrices in the perivascular space or from drug-releasing endovascular stents. Occlusive thrombosis, seen in 42% of control stent-bearing arteries, and partial thrombosis were virtually eliminated by heparin delivery from any route. Intimal area 14 days after balloon withdrawal denudation alone was reduced to an equal extent by continuous systemic heparin or by perivascular heparin for the first 3 days. In contrast, endovascular stents produced more exuberant neointimal hyperplasia, the inhibition of which required continuous rather than only early heparin administration. Neither perivascular delivery limited to the first 3 days nor stent-based delivery reduced neointimal hyperplasia as effectively. The antiproliferative and antithrombotic effects of heparin differ markedly, depending on the type of arterial injury and the mode of drug administration. Different forms of injury may require different therapies, and complications of arterial intervention such as excessive neointimal hyperplasia and thrombosis may demand alternate therapeutic regimens. Duration, dose, and site of delivery rather than frank resistance to therapy may explain why experimentally effective antiproliferative and antithrombotic agents fail clinically.

Activity assays of thiadiazine derivatives on Trichomonas vaginalis and amastigote forms of Trypanosoma cruzi.

Eight thiadiazine 1,1-dioxide derivatives were evaluated for antitrichomonal and antitrypanosomal activities. In vivo tests were performed on a murine model for trichomoniasis standardized in our laboratory. The capacity of compounds to clear visceral lesions in experimentally infected animals as well as their effects on the mortality time of mice were used as criteria for activity. One of the thiadiazines (compound 7b) showed an efficacy similar to that obtained with the reference drug metronidazole, although higher doses were required. Its toxicity on cell proliferation in tissue culture was moderate. In vitro assays on amastigote forms of Trypanosoma cruzi were carried out using cultures of Vero cells infected with metacyclic trypomastigotes. For one compound (1) trypanocidal activity resembled that of nifurtimox as assessed by microscopic counts of infected and uninfected cells. Unfortunately, this compound showed a high degree of cytotoxicity on Vero cell cultures.

Asparagine-linked oligosaccharides in malignant tumour growth

The expression of beta 1-6-branched complex-type oligosaccharides in several tumour cell models appears to be associated with enhanced metastatic potential. P2B, a major PHA-L-binding glycoprotein was isolated from metastatic MDAY-D2 cells and shown to bind to collagen, fibronectin and laminin with increased affinity after removal of N-linked sialic acid or polylactosamine. Sialylated polylactosamine-containing beta 1-6-branched oligosaccharides on proteins such as P2B and fibronectin may reduce cell adhesion and enhance tumour cell invasion. The loss of branched complex-type oligosaccharides in tumour cells due to somatic mutations or inhibition by swainsonine is also associated with decreased cell proliferation in tissue culture and slower rates of solid tumour growth in mice.

Non-neuronal cell proliferation in tissue culture: Implications for axonal regeneration in the central nervous system

A tissue culture model has been developed to examine the hypothesis that proliferating non-neuronal cells may constitute a physical and/or chemical barrier to regenerating neurons in the central nervous system. Explants from the sensorimotor cortex of 20-day-old fetal rats were cultured in serum medium (control) or serum medium containing 10(-5) M cytosine arabinoside (AraC), a mitotic inhibitor, for varying periods: 2-10, 4-12, 4-10, 4-8 and 4-7 days in vitro (DIV). The center and outgrowth zone of the explants were examined by phase-contrast microscopy at varying intervals between 3 and 18 DIV. The extent of central degeneration was greatest in explants treated with AraC from 2 DIV, and was least in the 4-7 day treated group in which only minimal degeneration was evident at 13 and 18 DIV. In the outgrowth zone at 18 DIV non-neuronal cell proliferation was controlled in the 4-10 day treated explants, although this was accompanied by extensive degeneration of neurites. Further examination of neurite viability, using a neurite viability ratio, revealed that degeneration was first evident at 6 DIV in the 2-10 day treated explants, but not until 9 or 13 DIV in any of the explants exposed to AraC from 4 days onwards. There was minimal degeneration in the 4-7 day treated explants. Electron microscopic examination revealed the presence of atypical inclusions in non-neuronal cells of 4-8 day treated explants, suggesting that the cytotoxic effect of AraC may be due to a disturbance in lipid and/or ganglioside metabolism. Quantitative electron microscopic analysis of the outgrowth zone at 18 DIV revealed a significant increase in the summated area of neuronal tissue (from 7 to 18 microns2/100 microns2) and a decline in the summated area of non-neuronal cells (from 83 to 61 microns2/100 microns2) for explants treated with AraC from 4 to 7 DIV compared to control. Diminishing the potential of non-neuronal cells to act as a barrier by controlling their proliferation may, therefore, be of importance in enhancing the regenerative response of central neurons.

On disturbance of homeostasis in organ-cultured tissue.

The intact membranous rat mesentery was cultured in Eagle's minimum essential medium containing no serum or only low concentrations of serum. The procedure is in some important respects superior to previous organ culture techniques. To estimate the extent of disturbance of homeostasis of the tissue in culture, the spontaneous mast-cell histamine release was quantitated after preculture preparation of the specimens and after different intervals in culture. Also, the proliferation of fibroblasts and mesothelial cells that predominate in the mesentery was assessed at 48 h by cytofluorometric quantitation of DNA in single-tissue cells. Spontaneous histamine release was time dependent during cultivation, amounting to ca. 50% at 48 h, and was affected by the medium used for moistening the tissue before cultivation. Culturing also brought about great spontaneous increase in the proliferation of fibroblasts and mesothelial cells, the rate being related to the concentration of serum. Addition of the mast-cell secretagogues 48/80 or polymyxin B at 1 h caused rapid release of 50 to 60% of the histamine and was followed by augmented proliferation in the serum-containing media. The spontaneous increase of cell proliferation in tissue culture may be causally related to mast-cell secretion. Further studies are needed to define factors influencing the spontaneous mast-cell secretion and the mast-cell-dependent mitogenesis in normal tissue cells.

Intrinsic tendon cell proliferation in tissue culture

Intrinsic tendon cell proliferation in tissue culture

Intrinsic tendon cell proliferation in tissue culture

This study examines the potential of tendon cells to participate in tendon repair. Explants were prepared from chicken sublimis tendons and studied in tissue culture. Under defined culture conditions, in the absence of tendon sheath, tendon cells were observed to migrate in a limited concentric fashion around the margin of a 2 mm window in the explant. When a plasma clot was present in the window, tendon cell migration and proliferation were accelerated and continued until the window was confluent with cells. Electron micrographs demonstrated that these tendon cells were fibroblasts associated with extracellular collagen fibrils. Specific biochemical analyses confirmed that these tendon fibroblasts were synthesizing collagen. This in vitro study demonstrates that tendons contain active fibroblasts capable of proliferation, migration, and collagen synthesis. These findings support the hypothesis that tendons can heal by intrinsic processes.

Effect of dilantin sodium analogues on cell proliferation in tissue culture.

SummarySeveral chemical analogues and chemically related compounds of Dilantin Sodium have been used to study and clarify the cell stimulatory effects of this compound. Two analogues (1 allyl, 5 ph...

Regeneration of axons in the vertebrate central nervous system.

Regeneration of axons in the vertebrate central nervous system.