The mammalian cell cycle in normal and abnormal growth

Cell division, a complex array of intracellular events, occurs in a highly ordered and carefully coordinated manner. This regulation is achieved by the sequential activation and deactivation of the members of a family of serine-threonine-specific protein kinases that consist of regulatory and enzymatic subunits, the cyclins and cyclin-dependent kinases. These enzymes, in turn, regulate the activity of other proteins involved in the mitogenic pathway. Mutations in the components of the regulatory pathways can lead to aberrant growth, including malignancies.     

Effectiveness of postoperative irradiation in stage IIIA non-small cell lung cancer according to regression tree analyses of recurrence risks

BACKGROUND: In the setting of grossly resected stage IIIA (N2 involvement) non-small cell lung carcinoma, the role of adjuvant postoperative thoracic radiation therapy (TRT) remains controversial. This study was initiated to subcategorize these patients into high-, intermediate-, and low-risk groups with respect to local recurrence and survival rates, and to determine whether there were certain subgroups of patients who were particularly likely or unlikely to benefit from postoperative TRT. METHODS: Two hundred twenty-four patients were studied. A regression tree analysis was used to separate patients who had undergone operation alone into groups that had a high, intermediate, or low risk of local recurrence and death. The effect of adjuvant postoperative TRT then was examined in each of these groups. RESULTS: The use of adjuvant postoperative TRT (compared with operation alone) was associated with an improvement in freedom from local recurrence and survival for patients who had an intermediate or high risk of local recurrence and death. However, the greatest level of improvement in freedom from local recurrence (p < 0.0001) and survival (p = 0.0002) associated with the use of adjuvant postoperative TRT was in the high-risk group. Similarly, but of lesser magnitude, the intermediate-risk group had improved freedom from local recurrence and survival rates with the use of adjuvant post-operative TRT (p = 0.002 and p = 0.01, respectively). For the low-risk group, the freedom from local recurrence and survival rates were not statistically different between the patients who received adjuvant postoperative TRT and those who underwent observation. CONCLUSIONS: Patients with non-small cell lung carcinoma involving ipsilateral mediastinal lymph nodes (stage IIIA) who undergo gross resection and who are at either high or intermediate risk for local recurrence and death are likely to benefit from adjuvant postoperative irradiation. The role of radiation therapy in low-risk patients is unclear. Prospective confirmation of these observations is warranted.     

Protective effects of glutathione isopropyl ester on the sensitivity of cultured cells to UVB irradiation

The effect of glutathione (GSH) isopropyl ester on cellular sensitivity to UVB irradiation was investigated in HeLaS3 cells. Pretreatment with 0.1-0.5 mM GSH isopropyl ester for 4 h significantly inhibited the decrease of thymidine (TdR) incorporation caused by UVB irradiation at a dose of 500 J/m2, whereas pretreatment with a high dose (1 mM) had no effect. The colony formation ability of the pretreated cells (0.3 mM) was significantly better than that of cells that received irradiation only. When the cells were treated with GSH isopropyl ester, their intracellular GSH level increased dose-dependently over a 4 h period, suggesting that GSH isopropyl ester was transported into the cells and there converted to GSH. Within 2 min of exposure, the intracellular GSH level depleted rapidly to about 75% of that in non-irradiated normal cells. In contrast, the GSH level in cells pretreated with 0.3 mM GSH isopropyl ester was maintained at the same level as that in normal cells, indicating that the maintenance of intracellular GSH level is due to converted GSH from GSH isopropyl ester. These results clearly show that intracellular GSH is involved in cell protection against photodamage, and that GSH isopropyl ester is a useful antioxidant for protection against photooxidative injury.     

Effect of aging on the sensitivity of growth hormone secretion to insulin-like growth factor-I negative feedback

To determine the effect of aging on the suppression of GH secretion by insulin-like growth factor (IGF)-I, we studied 11 healthy young adults (6 men, 5 women, mean +/- SD: 25.2 +/- 4.6 yr old; body mass index 23.7 +/- 1.8 kg/m2) and 11 older adults (6 men, 5 women, 69.5 +/- 5.8 yr old; body mass index 24.2 +/- 2.5 kg/m2). Saline (control) or recombinant human IGF-I (rhIGF-I) (2 h baseline then, in sequence, 2.5 h each of 1, 3, and 10 micrograms/kg.h) was infused iv during the last 9.5 h of a 40.5-h fast; serum glucose was clamped within 15% of baseline. Baseline serum GH concentrations (mean +/- SE: 3.3 +/- 0.7 vs. 1.9 +/- 0.5 micrograms/L, P = 0.02) and total IGF-I concentrations (219 +/- 15 vs. 103 +/- 19 micrograms/L, P < 0.01) were higher in the younger subjects. In both age groups, GH concentrations were significantly decreased by 3 and 10 micrograms/kg.h, but not by 1 microgram/kg.h rhIGF-I. The absolute decrease in GH concentrations was greater in young than in older subjects during the 3 and 10 micrograms/kg.h rhIGF-I infusion periods, but both young and older subjects suppressed to a similar GH level during the last hour of the rhIGF-I infusion (0.78 +/- 0.24 microgram/L and 0.61 +/- 0.16 microgram/L, respectively). The older subjects had a greater increase above baseline in serum concentrations of both total (306 +/- 24 vs. 244 +/- 14 micrograms/L, P = 0.04) and free IGF-I (8.5 +/- 1.4 vs. 4.2 +/- 0.6 micrograms/L, P = 0.01) than the young subjects during rhIGF-I infusion, and their GH suppression expressed in relation to increases in both total and free serum IGF-I concentrations was significantly less than in the young subjects. We conclude that the ability of exogenous rhIGF-I to suppress serum GH concentrations declines with increasing age. This suggests that increased sensitivity to endogenous IGF-I negative feedback is not a cause of the decline in GH secretion that occurs with aging.     

Haemopoietic growth factors, the cell cycle of acute myeloblastic leukaemia progenitors and sensitivity to cytosine arabinoside

We describe an 'in vitro' model which permits assessment of acute myeloblastic leukaemia (AML) progenitor cells' response to Ara-C alone and in conjunction with recombinant human (rh) cytokines by evaluating cell cycle characteristics of purified AML blast progenitors and their chemosensitivity in clonogenic culture before and after cytokine priming. Parallel investigation of Ara-C/cytokine treatment on normal CFU-GM progenitors was included as these cells are important for post-therapeutic reconstitution of haemopoiesis. Kinetic and clonogenic findings for AML marrows were extremely variable with G+GM-CSF or IL-3 priming. However, inclusion of rhSCF and resultant synergism with the other cytokines, introduced a pronounced element of consistency in AML results. Normal CFU-GM responded to rhG+GM-CSF or IL-3 with increased kinetic activity and sensitivity to Ara-C. rhSCF synergised strongly with the other factors, causing increased cell cycling and attainment of maximal chemosensitivity 'in vitro'. No correlation was evident between 'in vitro' findings for AML samples, patient FAB types or clinical outcome. This study highlights the fact that both normal and AML cells can be targeted by rh cytokines, particularly when rhSCF is included in priming cocktails.     

Differential sensitivity of human neuroblastoma cell lines to ethanol: correlations with their proliferative responses to mitogenic growth factors and expression of growth factor receptors

Early ethanol exposure depletes neurons in the developing nervous system, however the effects on neuronal precursors are not homogeneous. Some cells are more susceptible to ethanol toxicity than others. Growth factors are important mitogens for neuronal precursors. We tested the hypothesis that the differential sensitivity of neuronal precursors to ethanol is determined by their responses to growth factors using an in vitro model (SH-SY5Y, SK-N-SH, and IMR32 neuroblastoma cells) of neuronal precursors. The three cell lines were raised in a medium containing 10% or 0% fetal calf serum. Cells were exposed to ethanol and/or a growth factor. These factors included basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-I, nerve growth factor, and platelet-derived growth factors AA and BB. The numbers of cells per culture were counted both before and after 3 days of ethanol and/or growth factor treatment. In addition, the effect of ethanol exposure on the expression of receptors for these growth factors was examined. Neuroblastoma cells displayed differential sensitivity to ethanol. The growth of SH-SY5Y and SK-N-SH cells was inhibited by ethanol in a concentration-dependent manner. Ethanol did not affect cell viability. Thus, this inhibition resulted from a reduction of cell proliferation. In contrast, IMR32 cells were not affected by ethanol (even at concentrations as high as 800 mg/dl). The response to growth factors was also heterogeneous. In serum-supplemented medium, SH-SY5Y and SK-N-SH cells were stimulated by all of the tested growth factors. For cells raised in a serum-free medium, only the nerve growth factor was ineffective. IMR32 cells, however, were unaffected by most of these growth factors, regardless of the medium conditions. Ethanol blocked the action of all growth factors tested. In general, all cells expressed the specific receptors for the six growth factors. Only the expression of the basic fibroblast growth factor, insulin-like growth factor-I, and nerve growth factor receptors were reduced by ethanol exposure. In summary, neuroblastoma cells exhibit differential susceptibility to ethanol, and this correlates with their response to mitogenic growth factors. Some growth factors are a target of ethanol toxicity. These heterogeneous effects seem to parallel ethanol-induced changes of proliferating neuronal precursors in vivo.     

Inhibition of mammalian topoisomerase I by 1-nitro-9-aminoacridines. Dependence on thiol activation

It has been suggested that the rate of CD4 cell decline accelerates in parallel with decreasing numbers of cells; however, the statistical literature suggests the opposite. CD4 cells were counted about every 6 months in a cohort of 1264 human immunodeficiency virus-infected subjects (the Italian Seroconversion Study cohort). Kaplan-Meier analysis was used to estimate the time for CD4 cells to decline by 100 cells/mm3, conditional on reaching predefined levels. In addition, CD4 cell counts were modeled as a function of time since seroconversion in individuals with > or = 5 counts. Kaplan-Meier survival times for a 100 cell/mm3 decrease in CD4 cells increased as lower counts were reached (log rank test, P < .001). The shape of the overall fitted curve of the CD4 cell counts does not suggest an increasing rate of decline. Data from the Italian Seroconversion Study cohort do not show a general tendency for accelerating CD4 cell decline in association with lower counts.     

Targeting bacteriophage to mammalian cell surface receptors for gene delivery

Filamentous bacteriophages represent one of nature's most elegant ways of packaging and delivering DNA. In an effort to develop novel methods for ligand discovery via phage gene delivery, we conferred mammalian cell tropism to filamentous bacteriophages by attaching basic fibroblast growth factor (FGF2), transferrin, or epidermal growth factor (EGF) to their coat proteins and measuring CMV promoter-driven reporter gene expression in target cells. In this system, FGF2 was a more effective targeting agent than transferrin or EGF. The detection of green fluorescent protein (GFP) or beta-galactosidase (beta-Gal) activity in cells required FGF2 targeting and was phage concentration dependent. Specificity of the targeting for high-affinity FGF receptors was demonstrated by competing the targeted phage with FGF2, by the failure of FGF2-targeted bacteriophage to transduce high-affinity FGF receptor-negative cells, and by their ability to transduce these same cells when stably transfected with FGFR1, a high-affinity FGF receptor. Long-term transgene expression was established by selecting colonies for G418 resistance, suggesting that with the appropriate targeted tropism, filamentous bacteriophage can serve as a vehicle for targeted gene delivery to mammalian cells.     

Modulation of skin cell functions by transforming growth factor-beta1 and ACTH after ultraviolet irradiation

A new method was developed for binding poly-(ethylene oxide) (PEO) to polymer surfaces that involves the use of electron beam irradiation in two steps. In the first, methacrylic acid was grafted and polymerized to a polymer surface, changing it from hydrophobic to hydrophilic. Exposure of this surface to aqueous PEO solutions resulted in strong hydrogen bonding of the PEO, which was covalently grafted in a second radiation step. The PEO grafts were stable; they could not be removed with extensive washing with water, soaking in basic solution, or gentle mechanical scraping. Both monolayers and multilayers of PEO were formed. The density of the monolayers were found to have little dependence on the molecular weight or concentration of the PEO solution; multilayers could be controlled by varying the viscosity of the PEO solution and the method of application. The PEO-grafted monolayers were tested for their ability to prevent protein adsorption of cytochrome-c, albumin, and fibronectin. Monolayers of star PEO were the most effective, at best showing a 60% decrease in adsorption from untreated controls. One million molecular wight linear PEO monolayers were almost as effective as star monolayers, and 35,000 g/mol linear PEO was bound too closely to the surface, owing to its small size, to have much impact in preventing protein adsorption. The reason for the continued protein adsorption was believed to be due to a close grafting of the PEO to the surface, as well as the grafted methacrylic acid chains being long enough to extend through the PEO monolayer, thus being accessible on the surface.     

Dependence of mesenchymal cell responses on duration of exposure to bone morphogenetic protein-2 in vitro

Murine Leydig (TM3) and Sertoli (TM4) cell lines were studied as nonprofessional antigen-presenting cells using the antigen model of human choriogonadotropin (hCG alpha/beta) and specific T-cell hybridomas. Both cell lines were treated with IFN-gamma to induce I-A(d) and I-E(d) molecules expression. Only the TM3 cell line, which expressed MHC-class II molecules upon IFN-gamma stimulation, was able to uptake, process, and present the human choriogonadotropin beta subunit to related T-cell hybridomas. Interestingly, the TM3 cell line was incapable of presenting the human choriogonadotropin alpha subunit, the presentation of which, by classical APC, is highly efficient. Using T-cell hybridomas directed against the immunogenic regions of hCG alpha/beta previously described in BALB/c mice, we showed that the TM3 cell line generated a narrower peptide repertoire than classical APC (i.e., B cells, macrophages, and dendritic cells). This experimental system suggests that Leydig cells could initiate, in vivo, an autoimmune process directed against gonadal tissues. In particular, such a mechanism has been evoked in experimental autoimmune orchitis.     

Effects of glial cell line-derived neurotrophic factor on axonal growth and apoptosis in adult mammalian sensory neurons in vitro

The effects of glial cell line-derived neurotrophic factor on axonal outgrowth and apoptosis were studied in vitro using explanted dorsal root ganglia-peripheral nerve preparations of adult mice. In gels of matrigel or collagen type 1, glial cell line-derived neurotrophic factor increased both the numbers and lengths of axons growing out of explanted preparations, although less effectively than nerve growth factor. Stimulation of axonal outgrowth by glial cell line-derived neurotrophic factor was unaffected by K252a, a protein kinase inhibitor which blocks the effects of nerve growth factor and other neurotrophins acting through trk receptors. To determine the phenotype of the axons responding to glial cell line-derived neurotrophic factor, preparations were stained using antibodies to trkA, calcitonin gene-related peptide, 200,000 mol. wt phosphorylated neurofilaments (monoclonal antibody RT97) and the lectin Bandeiraea simplicifolia 1B4. RT97 recognizes large diameter neurons whilst 1B4 labels small diameter neurons which broadly do not express neurotrophin receptors. In preparations cultured with glial cell line-derived neurotrophic factor, significant increases in the numbers of outgrowing axons labelled with RT97 and 1B4 were observed but the numbers of calcitonin gene-related peptide-positive axons were not significantly increased and their staining intensity was generally faint. In separate preparations it was found that in the presence of glial cell line-derived neurotrophic factor, the majority of the 1B4 labelled axons were trkA negative, indicating that this factor can stimulate axonal growth in this population of neurons which do not respond to the neurotrophins. Spontaneous apoptosis in neurons and satellite cells occurs in explanted preparations of the type used in the present investigations, but in cryostat sections of preparations cultured in the presence of glial cell line-derived neurotrophic factor, the incidence of apoptosis was lower than in control preparations which had been cultured in the absence of this factor. This suggests that glial cell line-derived neurotrophic factor may promote survival of some adult sensory neurons in vitro.     

Dependence of microwave produced heating of cell suspensions on their concentration

This study compared three radionuclide techniques in distinguishing musculoskeletal infection from noninfectious inflammation. METHODS: Thirty-five orthopedic patients with suspected musculoskeletal infection were examined using three radionuclide techniques in sequence: triphasic bone scintigraphy, 99mTc radioleukocytes (99mTc-WBC) scintigraphy and 99mTc human immunoglobulin (99mTc-Hig) scintigraphy. Two "early" and "late" acquisitions were performed, at 4-6 hr and 20-24 hr postinjection, respectively. Patients who were diagnosed as suffering from noninflammatory lesions became the controls. We calcu"late"d for all studies one index of inflammation (Infl) as the ratio between counts in the uptake area and counts in an equal area of normal tissue. RESULTS: The "early" radiolabeled leukocytes and "late" Hig scintigraphy allowed the greatest ability to distinguish between infections and noninfectious inflammations (p < 0.011 and p < 0.016) with a sensitivity of 96.6% and 96.5% and specificity of 71% and 100%, respectively. Hig and radioleukocytes allowed distinguishing infections from noninflammatory diseases at both examinations. CONCLUSION: The "early" radioleukocyte scintigraphy allowed us to separate infections from noninfectious inflammations. In contrast, the same result can be obtained only with the "late" scan in the Hig study, but Hig mapped the spread of the inflammation into soft tissues better. Hig might be an alternative to radioleukocytes because of its simple preparation, similar accuracy and safety.     

Discrimination of mammalian growth hormones by peptide-mass mapping

Recognition by the legal authorities that growth hormones (GHs) may be abused to improve sporting performance and/or physique has led to the implementation of controls that make it an offence to produce, supply, possess or import and export GHs, with intent to supply, without the authority to do so. A method is described for the discriminatory analysis of human, equine, porcine and bovine GHs for forensic purposes. Peptide-mass mapping by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry following tryptic digestion gave sequence coverages of 97.4%, 93.7%, 94.2% and 90.6% for human, equine, porcine and bovine GHs respectively. The tryptic-mass maps generated were sufficient to discriminate between the four hormones analysed and thus provide unambiguous identification of each individual GH. Identification of the N-terminal peptides of recombinant equine and porcine GHs, which possess additional methionine residues, within the tryptic-mass maps may provide the basis of a test to indicate exogeneous administration rather than endogenous secretion of GH in performance dogs and horses.     

Genetic integrity of transforming growth factor beta (TGF-beta) receptors in cervical carcinoma cell lines: loss of growth sensitivity but conserved transcriptional response to TGF-beta

PURPOSE: Fractal kinetics was used for the analysis of the carrier-mediated transport for drugs across the intestinal epithelial cells. METHODS: The transport was examined under various agitation rates using a monolayer of Caco-2 cells and rabbit ileum sheets. RESULTS: The passive transport of antipyrine across Caco-2 cells was increased with the increasing rate of agitation and was supposed to be caused by a change in the thickness of the unstirred water layer. On the contrary, in the case of L-lactic acid transport, which follows a carrier-mediated transport mechanism, the more the agitation rate controlling the fractal dimension was increased, the more the permeability rate across the Caco-2 cells was decreased. Fractal kinetic analysis of L-lactic acid transport indicated that the permeability was caused by a single saturable process. Similar agitation effects with L-lactic acid transport were observed in the transport of phenylalanine and cephradine in Caco-2 cells. However, the permeability rates of benzoic acid and 3-O-methyl-D-glucose across Caco-2 cells and L-lactic acid transport across the rabbit ileum tissue indicated the maximum levels at a designated agitation rate. This phenomenon was likely to be caused by the agitation effects controlling not only the fractal environment but also the unstirred water layer. CONCLUSIONS: Carrier-mediated transports are well defined by fractal kinetics rather than classical kinetic analysis. Fractal kinetics are one of the important areas for understanding and confirming the properties of a carrier-mediated transport process.     

Microwave irradiation influences on the state of human cell nuclei

Changes of electrokinetic properties of cell nuclei and the quantity of granules of heterochromatin located near the nuclear envelope in nuclei of human buccal epithelium cells were studied under the influence of electromagnetic fields in vitro. Irradiation of cells was realized by means of a semiconductor generator of millimeter radiation (wavelength 7.1 mm, frequency 42.2 GHz), the Yav-1 apparatus for extremely high frequency therapy. It was shown that irradiation of cells induced a decrease in electric charge of native human buccal epithelium cell nuclei and an increase in chromatin condensation in nuclei. The observed effects depend on irradiation dose and individual peculiarities of donors.