EB荧光分析法测定肿瘤细胞DNA交联及增殖活性

应用EB荧光分析法测定体外培养人宫颈癌细胞株(HeLa)、人白血病细胞株(HL-60),增殖性和非增殖性人外周血淋巴细胞(PBL)的DNA含量及其交联度(DNA cross-link),并据此研究不同增殖状态细胞与其DNA百分交联度(ct%)的相互关系.结果显示,HeLa细胞、HL-60细胞、增殖性和非增殖性PBL的DNA ct%分别为36.5、 22.5、 20.2和0,表明不同增殖速度或周期的细胞具有不同的DNA交联反应,而非增殖性细胞或G₀期细胞不产生DNA交联反应.     

An epidermal proliferative unit-like structure in the epithelium of mouse bladder observed by backscattered electron imaging

Summary The simultaneous use of a silver-staining technique, backscattered electron imaging and stereo-tilts has made it possible to visualize the spatial distribution of cell nuclei in the stretched epithelium of the bladder of mice. This study has led to the observation that a structural organization resembling the epidermal proliferative unit, previously found in the skin exists also in bladder epithelium. However, the proliferative unit in the bladder was different in that it contained a higher number of cells per unit, and an absence of columns of inactive squamous cells. These findings may indicate that epidermal proliferative unit-like structures represent a common form of organization in some epithelia.     

Karyological analysis of hybridoma cells after prolonged cultivation

The karyological properties, the level of monoclonal antibody production and the proliferative properties of hybridoma strains after their prolonged passage in vivo and in vitro have been studied. Hybridoma EKO-G-2 having the supertetraploid set of chromosomes has proved to be a more stable antibody producer and to possess better proliferative properties. The suggestion has been made that the stability of antibody production is linked with the surplus number of chromosomal copies.     

The proliferative potential of hematopoietic stem cells (CFUs) during serial passage in irradiated mice as foci of ectopic hematopoiesis

During serial passages of hemopoietic tissue as a whole ectopic hemopoietic site under renal capsule of irradiated mice gradual decrease of proliferative potential of CFUs has been observed. The results suggest that the most probable cause of such decline is random loss of precursor cells with high but limited proliferative potential during serial passages of hemopoietic tissue.     

Kinetic pattern of human urinary transitional cell carcinoma

The potential proliferative activity, expressed as labelling index, has been studied in 24 cases of bladder transitional cell carcinomas. The data showed a wide range of values, varying between 0.4 and 36%. The average proliferative activity was 3.2%. An analysis of the connection between the labelling index and the histological grading has also brought out the existence of a gradient of proliferative activity; average values of 1.6-2.53% and 3.49%, respectively, were found for grades I-II and grade III.     

Accurate Determination of S-Phase Fraction in Proliferative Cells by Dual Fluorescence and Peroxidase Immunohistochemistry with 5-Bromo-2��-Deoxyuridine (BrdU) and Ki67 Antibodies

To ensure the maintenance of tissues in mammals, cell loss must be balanced with cell production, the proliferative activity being different from tissue to tissue. In this article, the authors propose a new method for the quantification of the proliferative activity, defined as the S-phase fraction of actively cycling cells, by dual labeling with fluorescence and peroxidase immunohistochemistry using BrdU (marker of S-phase) and Ki67 antibodies (marker of G₁-, S-, G₂-, and M-phases) after a one-step antigen retrieval. In the generative cell zones of fundic and pyloric glandular stomachs, where the majority of cells were cycling, the authors measured a proliferative activity of 31%. In the epithelium of the forestomach and the skin, where cycling cells are intermingled with G₀ and differentiated cells, proliferative activities were 21% and 13%, respectively. In the adrenal cortex, in which cycling cells were sparsely distributed, the proliferative activity reached 32%. During the regenerative process in the skin after a lesion, the proliferative activity increased in proximity to the wound. The present one-step dual-labeling method has revealed that the proliferative activity is different between tissues and depends on the physiological or pathological state.     

Blockade of physiologically secreted IFN-gamma inhibits human T lymphocyte and natural killer cell activation

The role of physiologically secreted human IFN-gamma in T lymphocyte and NK cell activation has been probed with a panel of mouse mAb directed against various epitopes of the human IFN-gamma molecule, or human IFN-gamma R. Addition to the culture medium of those mAb that neutralize the antiviral activity of IFN-gamma or interact with its receptor inhibited proliferative and cytotoxic responses elicited in PBL by HLA alloantigens, anti-CD3 mAb, and IL-2, but not the proliferative response to PHA. The IFN-gamma blockade also inhibited IFN-gamma, IL-2, and TNF-alpha release during MLC. Kinetic experiments showed that reduction of proliferative and cytotoxic responses to HLA alloantigens is maximal when IFN-gamma is blocked within the first 48 h. Exogenous rIFN-gamma restored the proliferative response only when added at the beginning. Moreover, when IFN-gamma was blocked, T lymphocytes recovered from 6-day MLC displayed a profound decrease in their expression of p55 and p75 chains of the IL-2R, as well as in the number of high-affinity IL-2 binding sites. These findings strongly suggest that IFN-gamma is required in the early phases of induction of the oligo- and polyclonal proliferative and cytotoxic responses of lymphocytes.     

Comparative study of the immunosuppressive properties of trophoblastic beta 1-glycoprotein and placental alpha 2-microglobulin in vitro

It has been shown that trophoblastic beta 1-glycoprotein (TBG) and placental alpha 2-microglobulin (PAMG-2) in concentrations 60-120 micrograms/ml suppresses both the inductive and proliferative phase of unidirectional mixed lymphocyte reaction in mice, as well as proliferative responses to phytohemagglutinin or pokeweed mitogen. TBG protein was more effective. The proteins were not toxic for lymphocytes.     

Proliferative response of peripheral blood lymphocytes to mitogens in the owl monkey Aotus nancymae

The new world primate Aotus sp. has been recommended by the World Health Organization as a model for evaluation of malaria vaccine candidates, given its susceptibility to experimental infection with the human malaria parasites Plasmodium falciparum and P. vivax. The present study examined the in vitro proliferative response of peripheral blood mononuclear cells (PBMCs) isolated from Aotus monkeys, utilizing a wide range of mitogens. Results presented herein demonstrate that the in vitro proliferative response of PBMCs from the Aotus sp. is quite variable from monkey to monkey for each of the mitogens assessed. PBMCs from the Aotus monkey exhibited a delayed kinetic proliferative response and, particularly, a different sensitivity to proliferation in response to various concentrations of Phytohemagglutinin-P and favin lectins, the phorbol ester Phorbol myristate acetate and the calcium ionophore ionomycin. Altogether, our findings are consistent with the conclusion that the in vitro proliferative response of PBMCs from the Aotus differ in their activation requirements compared with PBMCs from humans.     

Intraocular injection of an aptamer that binds PDGF-B: a potential treatment for proliferative retinopathies

Platelet-derived growth factor-B (PDGF-B) has been implicated in the pathogenesis of proliferative retinopathies and other scarring disorders in the eye. In this study, we sought to test the therapeutic potential of an aptamer that selectively binds PDGF-B, ARC126, and its PEGylated derivative, ARC127. Both ARC126 and ARC127 blocked PDGF-B-induced proliferation of cultured fibroblasts with an IC50 of 4 nM. Pharmacokinetic studies in rabbits showed similar peak vitreous concentrations of approximately 110 microM after intravitreous injection of 1 mg of either ARC126 or ARC127, but the terminal half-life was longer for ARC127 (98 versus 43 h). Efficacy was tested in rho/PDGF-B transgenic mice that express PDGF-B in photoreceptors and develop severe proliferative retinopathy resulting in retinal detachment. Compared to eyes injected with 20 microg of scrambled aptamer in which five of six developed detachments (three total and two partial), eyes injected with ARC126 (no detachment in five of six and one partial detachment), or ARC127 (no detachment in six of six) had significantly fewer retinal detachments. They also showed a significant reduction in epiretinal membrane formation. These data demonstrate that a single intravitreous injection of an aptamer that specifically binds PDGF-B is able to significantly reduce epiretinal membrane formation and retinal detachment in rho/PDGF-B mice. These striking effects in an aggressive model of proliferative retinopathy suggest that ARC126 and ARC127 should be considered for treatment of diseases in which PDGF-B has been implicated, including ischemic retinopathies such as proliferative diabetic retinopathy, proliferative vitreoretinopathy (PVR), and choroidal neovascularization.     

Hepatoma-derived growth factor. Significance of amino acid residues 81-100 in cell surface interaction and proliferative activity

Hepatoma-derived growth factor (HDGF) has proliferative, angiogenic, and neurotrophic activity. It plays a putative role in the development and progression of cancer. When expressed in cells, the mitogenic activity of HDGF depends on its nuclear localization, but it also stimulates proliferation when added to the cell culture medium. A cell surface receptor for HDGF has not been identified so far. We investigated the interaction of various purified recombinant HDGF fusion proteins with the cell surface of NIH 3T3 fibroblasts. We showed that binding of a HDGF-beta-galactosidase fusion protein to the cell surface of NIH 3T3 fibroblasts was saturable, occurred with high affinity (K(D) = 14 nm), and had a proliferative effect. We identified a peptide comprising amino acid residues 81-100 within the amino-terminal part of HDGF that bound to the cell surface of NIH 3T3 cells with saturation and affinity values similar to those of HDGF. When added to primary human fibroblasts, this peptide stimulated proliferation. Substitution of a single amino acid (K96A) within this peptide was sufficient to abolish its binding to the cell surface and its proliferative activity. In contrast, when expressed transiently in NIH 3T3 cells, a HDGF-beta-galactosidase fusion protein in which amino acid residues 81-100 were deleted still had proliferative activity, whereas a fusion protein containing only the 81-100 peptide did not. Our results suggest the existence of a plasma membrane-located HDGF receptor for which signaling depends on amino acid residues 81-100 of HDGF. This region differs from the one that has been recently identified to be essential for mitogenic activity depending on the nuclear localization of HDGF. Thus, HDGF exerts its proliferative activity via two different pathways.     

Cell-intrinsic regulators of proliferation in vertebrate retinal progenitors

The proliferative expansion of retinal progenitor cells (RPCs) is a fundamental mechanism of growth during vertebrate retinal development. Over the past couple of years, significant progress has been made in identifying genes expressed in RPCs that are essential for their proliferation, and the molecular mechanisms are beginning to be resolved. In this review, we highlight recent studies that have identified regulatory components of the RPC cell cycle machinery and implicate a set of homeobox genes as key regulators of proliferative expansion in the retina.     

A proliferative melanoma cell phenotype is responsive to RAF/MEK inhibition independent of BRAF mutation status

Oncogenic mutations within the MAPK pathway are frequent in melanoma, and targeting of MAPK signaling has yielded spectacular responses in a significant number of patients that last for several months before relapsing. We investigated the effects of two different inhibitors of MAPK signaling in proliferative and invasive melanoma cell cultures with various mutations in the MAPK pathway. Proliferative melanoma cells were more susceptible to pathway inhibition than invasive phenotype cells, irrespective of BRAF mutation status, while invasive phenotype cell response was dependent on BRAF mutation status. Critically, MAPK pathway inhibition of proliferative phenotype cells resulted in acquisition of invasive phenotype characteristics. These results show that melanoma cell phenotype is an important factor in MAPK pathway inhibition response. This suggests that while current therapeutic strategies target proliferative melanoma cells, future approaches should also account for the invasive phenotype population.     

Trapping of growth factors by catechins: a possible therapeutical target for prevention of proliferative diseases

The prevention of cancer through dietary intervention is currently receiving considerable attention. Several epidemiological studies substantiate that green tea has a protective effect against a variety of malignant proliferative disorders such as lung cancer, breast cancer and prostate cancer. This preventive potential of green tea against cancer is attributed to the biologically active flavonoids called catechins. Epigallocatechin 3-o-gallate, the major catechin found in green tea, mediates diverse physiological and pharmacological actions in bringing about the regression of the tumors and also lowers the risk of nonmalignant cardiovascular proliferative diseases. Much of the current research is being focused on how these catechins specifically bring about the regression of the experimentally induced tumors both in vitro and in vivo. These catechins exert diverse physiological effects against proliferative diseases by several mechanisms, most of which are not completely characterized. This review summarizes the mechanisms by which these catechins play an essential role in regulating the process of carcinogenesis, with a special emphasis on how these catechins antagonize the growth factor-induced proliferative disorders.     

TESTOSTERONE-INDUCED CELL PROLIFERATION IN THE ACCESSORY SEX GLANDS OF MICE AT VARIOUS TIMES AFTER CASTRATION

The proliferative response to testosterone in the accessory sex glands (seminal vesicle and coagulating gland) of castrated male Balb/c mice has been examined by pulse and continuous thymidine-labelling experiments, and by the fraction of labelled mitoses technique. Progressive reductions in cellularity followed castration, and by varying the time elapsing between castration and the initiation of testosterone treatment, it was clear that the size of the response depended upon the number of cells in the tissue, relative to the normal complement. Interpretation of FLM data was difficult in periods where proliferative rates changed rapidly. We have attempted to explain the cell kinetic events by postulating a G₀ compartment, from which cells are stimulated to enter the proliferative cycle before subsequently returning to an out of cycle state. It was thought unlikely that substantial changes in cell cycle time occurred. In both the accessory sex glands, the overall form of the continuous thymidine labelling curves showed that most proliferative cells entered DNA synthesis in a shorter time after stimulation at 14 days after castration than they did at 3 days after castration. The data were not consistent with cells moving deeper into G₀ with time after castration. In the seminal vesicle almost all epithelial cells were potentially proliferative by 3 days after castration. In the coagulating gland only 30% were potentially proliferative at 3 days, increasing to 85% at 14 days after castration. However, such proportional increases represented much smaller changes in terms of absolute numbers of cells, because of a concomitant decline in cellularity from 3 to 14 days after castration.     

Testosterone-induced cell proliferation in the accessory sex glands of mice at various times after castration

The proliferative response to testosterone in the accessory sex glands (seminal vesicle and coagulating gland) of castrated pale Balb/c mice has been examined by pulse and continuous thymidine-labelling experiments, and by the fraction of labelled mitoses technique. Progressive reductions in cellularity followed castration, and by varying the time elapsing between castration and the initiation of testosterone treatment, it was clear that the size of the response depended upon the number of cells in the tissue, relative to the normal complement. Interpretation of FLM data was difficult in periods where proliferative rates changed rapidly. We have attempted to explain the cell kinetic events by postulating a G0 compartment, form which cells are stimulated to enter the proliferative cycle before subsequently returning to an out of cycle state. It was thought unlikely that substantial changes in cell cycle time occurred. In both the accessory sex glands, the overall form of the continuous thymidine labelling curves showed that most proliferative cells entered DNA synthesis in a shorter time after stimulation at 14 days after castration than they did at 3 days after castration. The data were not consistent with cells moving deeper into G0 with time after castration. In the seminal vesicle almost all epithelial cells were potentially proliferative by 3 days after castration. In the coagulating gland only 30% were potentially proliferative at 3 days, increasing to 85% at 14 days after castration. However, such proportional increases represented much smaller changes in terms of absolute numbers of cells, because of a concomitant decline in cellularity from 3 to 14 days after castration.     

Effect of beta-endorphin and myelopeptides on the cAMP level and proliferation of lymphocytes in vitro

Beta-endorphin (10(-11)-10(-9) M) has been shown to induce naloxone-independent depression of the proliferative activity of human peripheral lymphocytes (HL), stimulated by pokeweed mitogen without affecting PHA-stimulated HL proliferation. Beta-endorphin (10(-10)-10(-7) M) also caused changes in HL cAMP level, that were blocked by naloxone. Marked individual sensitivity to beta-endorphin effects has been noted. It has been also shown that a bone marrow preparation, stimulating antibody production (myelopeptides), causes naloxone-independent depression in the proliferative activity of HL, stimulated by PHA and pokeweed mitogen, as well as naloxone-blocked decrease in cAMP HL level. It has been concluded that beta-endorphin interacts with several types of opiate lymphocyte receptors and that opioids, contained in myelopeptides, are involved in the realization of myelopeptide effect on lymphocytes.     

Cell proliferation in the mouse parotid gland after unilateral parotidectomy.

Unilateral parotidectomy with or without total submandibulectomy has been used to induce cell proliferation in mouse parotid gland. Maximum DNA synthesis and mitosis were recorded four and five days after the operation. The double operation increased the proliferative response. Such proliferative stimulii was not accompanied by secretion and was sex independent. On the other hand, the response decreased with age. RNA and protein synthesis inhibitors showed that the stimulation of DNA synthesis depends on early protein synthesis, which seems to be synthesized on a preexisting template.