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PubMed2000

Activation of protein kinase Calpha inhibits growth of pancreatic cancer cells via p21(cip)-mediated G(1) arrest.

We have analyzed human pancreatic cancer cells to explore the growth regulatory function of protein kinase C (PKC)alpha. PKCalpha subcellular redistribution, activation kinetics and downregulation were examined in detail and correlated to immediate and delayed effects on cell-cycle regulatory pathways. TPA treatment resulted in transient PKC(&agr;) activation accompanied by translocation of the enzyme into membrane and nuclear compartments, and was followed by subsequent downregulation. TPA-induced inhibition of DNA synthesis was prevented by a PKC-antagonist and was reproduced by microinjection of recombinant PKCalpha, indicating that activation of this isoenzyme was required and sufficient for growth inhibitory effects. PKC(&agr;) activation arrested cells in the G(1) phase of the cell cycle as a consequence of selective inhibition of cyclin dependent kinase (CDK)2 activity with concomitant hypophosphorylation of Rb. The inhibition of CDK2 activity resulted from induction of p21(cip1) cyclin-dependent kinase inhibitors. Levels of p21(cip1) remained elevated and CDK2 activity repressed in spite of PKCalpha downregulation, indicating that downstream effectors of PKCalpha are the primary determinants for the duration of PKC-mediated growth inhibition. The PKCalpha-induced block in cell proliferation persisted even though cells were kept in the presence of growth factors, suggesting that induction of PKCalpha results in a permanent withdrawal of pancreatic cancer cells from the cell cycle.

Apoptosis↗
PubMed1994

Retroviral lineage studies of the rat olfactory epithelium.

Replication-incompetent retroviral vectors that encode the heritable marker enzyme, beta-galactosidase, were used to study the lineage relationships of cells in the olfactory epithelium of unmanipulated animals and in the olfactory epithelium as it reconstitutes after lesion. Virally-marked cells are categorized as to type based on their position in the epithelium and on expression of NCAM (limited to neurons) and the carbohydrate moiety recognized by Griffonia lectin (limited to the dark/horizontal basal cells and the microvillar class of supporting cells). Direct injections of the vectors into the olfactory epithelium of otherwise intact animals produce clusters of beta-galactosidase-labeled cells when assessed 6-10 days after infection; these clusters are composed of neurons and NCAM-negative/lectin-negative light/globose basal cells exclusively. In contrast, clusters of virally-marked cells after MeBr-induced lesion of the epithelium frequently contain both neurons and supporting cells, as well as both types of basal cells. Other clusters contain supporting cells and/or Bowman's gland/duct cells. It is likely that the clusters of marked cells are derived from a single founder cell, i.e. the cells are clonal and lineally related, since the clusters are widely dispersed. Furthermore, infusion of mixtures of viruses that can be distinguished on the basis of the type and subcellular localization of the marker enzyme that is expressed produce clusters that are homogenous with respect to enzyme type, providing strong evidence in favor of the notion that the clusters are clonal in nature. Thus, the founders of the clones that contain neurons, supporting cells and basal cells are pluripotent in their capacity for differentiation. It is unlikely that the pluripotent cells are found in Bowman's gland/duct, since we have yet to observe a clone that contains neurons and cells in Bowman's gland/duct. Hence, the pluripotent stem cells are to be found in the basal cell compartment of the epithelium. However, the exact nature of these stem cells remains unknown and a subject for future investigation.

Animals↗
PubMed1999

Spatial-specific TGF-beta1 adenoviral expression determines morphogenetic phenotypes in embryonic mouse lung.

The precise spatial-temporal role that expression and activation of transforming growth factor (TGF)-beta plays in mammalian organ morphogenesis remains incompletely understood. Using replication deficient adenoviral vectors containing engineered TGF-beta1 cDNAs, we studied the spatial effects of locally over-expressing either latent or mutated, constitutively active TGF-beta1 protein during embryonic mouse lung branching morphogenesis in culture. Transfer of exogenous genes into lung epithelium was achieved by intra-tracheal micro-injection of recombinant adenovirus, while submerging lungs in virus resulted in gene transfer into the pleura and subjacent mesenchymal cells, as revealed by cytochemical staining for beta-galactosidase. Only lungs transfected with active, but not latent TGF-beta1 gene, showed elevated levels of active TGF-beta. Epithelial over-expression of active, but not latent TGF-beta1, via intra-tracheal micro-injection inhibited lung branching morphogenesis by 36 %. In contrast, lungs submerged with either active or latent TGF-beta1 recombinant virus did not demonstrate an inhibitory effect upon branching. Pulmonary gene regulation was assayed by competitive polymerase chain reaction coupled with reverse transcription. Direct respiratory tract micro-injection of adenovirus over-expressing active TGF-beta1 resulted in a dose-dependent inhibition of epithelial surfactant protein (SP)-C and SP-B mRNA levels by up to 76 % and 70 %, respectively, while in contrast, fibronectin and matrix Gla protein (MGP) mRNA levels remained stable. However, lungs that had been submerged in adenovirus expressing active TGF-beta1 demonstrated a concentration-dependent induction of both fibronectin and MGP mRNA levels up to 4.3- and 4.7-fold respectively in the presence of 1 x 10(11) pfu/ml active TGF-beta1 virus. On the other hand, lungs treated with adenovirus expressing latent TGF-beta1 either by micro-injection or submerging failed to demonstrate any regulatory effect either upon epithelial or mesenchymal gene expression. We conclude that adenovector-mediated over-expression of activated TGF-beta1 in specific spatial compartments results respectively in either inhibition of branching morphogenesis and epithelium-specific gene expression, or in induction of matrix gene expression without affecting morphogenesis or epithelium-specific gene expression, depending on the route of administration. Also, the lack of effect of latent TGF-beta1 over-expression strongly suggests that TGF-beta activation per se provides an important locus of fine regulation of the spatial effects of TGF-beta signaling during embryonic lung branching morphogenesis.

Adenoviridae↗
PubMed1998

Mucosal HIV infection.

The gastrointestinal tract is not only a major site of clinical manifestations of the acquired immune deficiency syndrome but also an important compartment for HIV infection at all stages of the disease. In intestinal biopsies HIV has been detected so far mainly in mononuclear cells of the lamina propria but also in epithelial cells. Primary and permanent epithelial cell lines can be productively infected by HIV in vitro and epithelial cell monolayers can rapidly transport HIV by transcytosis. Both phenomena are most effective for cell-associated virus and do not require CD4 expression but seem to involve the interaction of gp120 with galactosyl ceramide. In vivo, productive HIV infection of epithelial cells appears to be rare if it occurs at all. Mucosal HIV infection is highly active at all stages of HIV infection and the gastrointestinal tract is probably a major source of HIV in the body. Highest mucosal HIV production is found rather early in HIV disease and is associated with histological abnormalities and gastrointestinal symptoms. The excessive production of HIV in the intestine appears to be due to transcriptional or translational upregulation and local variations in HIV production correlate with local cytokine levels.

Galactosylceramides↗
PubMed2003

Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice.

Inefficient gene transfer, inaccessibility of stem cell compartments, transient gene expression, and adverse immune and inflammatory reactions to vector and transgenic protein are major barriers to successful in vivo application of gene therapy for most genetic diseases. Prenatal gene therapy with integrating vectors may overcome these problems and prevent early irreparable organ damage. To this end, high-dose attenuated VSV-G pseudotyped equine infectious anaemia virus (EIAV) encoding beta-galactosidase under the CMV promoter was injected into the fetal circulation of immuno-competent MF1 mice. We saw prolonged, extensive gene expression in the liver, heart, brain and muscle, and to a lesser extent in the kidney and lung of postnatal mice. Progressive clustered hepatocyte staining suggests clonal expansion of cells stably transduced. We thus provide proof of principle for efficient gene delivery and persistent transgene expression after prenatal application of the EIAV vector and its potential for permanent correction of genetic diseases.

Animals↗
PubMed1983

Kinetics of telencephalic neural cell proliferation during the fetal regeneration period following a single X-irradiation at the late organogenesis stage. II. Cycle times and the size of the functional compartment of neural epithelial cells of distinct lesion districts.

Autoradiographic studies were conducted at the cerebral hemispheres of mouse embryos X-irradiated on day 12 of gestation and of normal litter mates during the subsequent developmental period. By counting the percentage of labeled mitoses the generation time, the potential doubling time, the growth fraction, as well as the length of the individual cell cycle stages of the neuroblast cells were determined. A continuous increase of generation time was found in the normal brains, concomitant with a latero-medial gradient in telencephalic wall differentiation progress. After X-irradiation this normal differentiation pattern still prevails, but with some marked topographical peculiarrities. The most important finding was a significant lengthening of the generation time at the medially situated rudiments of the ventricular zone and, similarly at the heterotopic cell islets located within the intermediary zone. Concomitant with this effect, which was seen mainly on days 15 and 17 of gestation, there was a marked increase of mitotic time of these special neuroblasts. The latter finding was regarded as a random event only, which has no causal relationship to the pathogenesis of the heterotopic islets or similar overgrowth anomalies after X-irradiation. In spite of the long generation time of these histological peculiarities, they make a considerable contribution to the regeneration of the injured telencephalic wall: Up to day 15 of gestation the heterotopias had a growth fraction of nearly 1.0 (= 100%), whereas the percentage of proliferating cells within the orthotopic remainders of the ventricular zone was only 44%.

Animals↗
PubMed2000

Establishment of latent HIV-1 infection of resting CD4(+) T lymphocytes does not require inactivation of Vpr.

The introduction of highly active antiretroviral therapy (HAART) for the treatment of HIV-1 infection has allowed dramatic reductions in plasma virus levels to below the limit of detection in many patients. However, latently infected CD4(+) memory T lymphocytes persist as an important reservoir for the virus in the presence of this aggressive therapy and represent a major barrier to HIV-1 eradication with HAART. The mechanism through which the latent compartment is formed has not yet been established. It may involve actively proliferating CD4(+) T-cell intermediates that are infected with HIV-1 and revert back to a resting state, carrying integrated provirus at some low frequency. The HIV-1 accessory protein Vpr, which mediates G(2) cell cycle arrest in host cells, may interfere with the formation of the latently infected T cells by preventing them from exiting the cell cycle to return to a resting state. To investigate the role of the Vpr in the formation of latently infected memory T cells, we cloned and characterized vpr genes from viruses in the latent reservoir. Both sequence analysis and functional assays demonstrated that the vpr gene products of the viruses isolated from the latent pool did not differ significantly from those of a functional Vpr (NL4-3). These results indicate that the generation of resting G(0) memory T lymphocytes that carry latent HIV-1 provirus occurs despite the G(2) arrest function of the vpr gene product.

Amino Acid Sequence↗
PubMed1975

The fine structure and cell kinetics of mouse epidermis after wounding.

A variable amount of cornified tissue removed from mouse dorsal epidermis results in stimulation of the entire basal layer. Stimulation does not appear to be dependent on damage to an indiviaual epidermal proliferative unit (EPU). The immediate reaction to wounding is a rapid movement of cells from the basal layer to the differentiating compartment resulting in depopulation of the basal layer, which is followed by a burst in DNA-synthetic activity. The result of the increased transit of cells through the epidermis is that various aspects of keratinization can appear abnormal. The Langerhans cells show several changes, often appearing suprabasal and becoming smaller, rounded cells with a less-clear cytoplasm and fewer granules. The initial migratory reaction results in a largely normal epidermis on the third day. This reaction is followed by a transient hyperplasia which reaches its peak on the sixth to seventh day and gradually returns to normal by the fourteenth to fifteenth day. The hyperplasia is characterized by a loss of the ordered stacking of cornified cells which become shorter and thicker than normal. There is a return to the stacked state beginning on the tenth day. The Langerhans frequency is apparently at its lowest on days 6-7 when the proliferation levels are at their maximum. An inverse relationship appears to exist between relative Langerhans cell frequency and cell proliferation rate. The data suggest that the frequency of Langerhans granules also changes during the course of the hyperplasia, peak levels being observed just before the decline in proliferative activity.

Animals↗
PubMed1998

A semiautomated fluorescence-based cell-to-cell fusion assay for gp120-gp41 and CD4 expressing cells.

A novel fluorescence-based method was developed to measure HIV envelope glycoprotein (env)-CD4-mediated cell fusion. This method measures the spread of a fluorescent dye as the cytosolic compartments of adjacent cells become contiguous upon cell-to-cell fusion. Calcein-labeled CD4+ Sup-T1 cells were seeded onto a monolayer of unlabeled TF228.1.16 cells, which stably express env, the gp120-gp41 complex. Changes in the following parameters were measured using a stage-scanning laser microscope: total fluorescent area, average fluorescent area, and average shape factor. Anti-CD4 monoclonal antibodies, anti-Leu3a, and OKT4E were shown to block fusion in a dose-dependent manner, while OKT4 had no effect. Aurin tricarboxylic acid, a compound that interferes with the binding of anti-Leu3a mAb and gp120 to CD4+ human peripheral blood lymphocytes, T20, a peptide that interferes with gp41, and cytochalasin D, a microfilament disrupter, all blocked fusion in a dose-dependent manner. This semiautomated assay can be used to quickly assess the effectiveness of compounds acting at different sites to block CD4 and env initiated cell-to-cell fusion.

Antibodies, Monoclonal↗
PubMed1991

Differences in the action and metabolism between retinol and retinoic acid in B lymphocytes.

We have previously reported on the dependency of activated B lymphocytes for retinol. Here we confirm and extend these findings that cells deprived of retinol perish in cell culture within days, displaying neither signs of apoptosis nor of cell cycle arrest. Cell death can be prevented by physiological concentrations of retinol and retinal, but not by retinoic acid or three synthetic retinoic acid analogues. To exclude the possibility that retinoic acid is so rapidly degraded as to escape detection, we have tested its stability in intra- and extracellular compartments. Contrary to expectation, we find that retinoic acid persists for longer (t 1/2 3 d) in cultures than retinol (t 1/2 1 d). Furthermore, despite the use of sensitive trace-labeling techniques, we cannot detect retinoic acid or 3,4-didehydroretinoic acid among retinol metabolites. However, retinol is converted into several new retinoids, one of which has the ability to sustain B cell growth in the absence of an external source of retinol, supporting the notion of a second retinol pathway. We have also determined which of the known retinoid-binding proteins are expressed in B lymphoblastoid cells. According to results obtained with polymerase chain reaction-assisted mRNA detection, they transcribe the genes for cellular retinol- and cellular retinoic acid-binding proteins, for the nuclear retinoic acid receptors, RAR-alpha, -gamma, and RXR-alpha, but not RAR-beta. Our findings that B cells do not synthesize retinoic acid or respond to exogenous retinoic acid on the one hand, but on the other hand convert retinol to a novel bioactive form of retinol, suggest the existence of a second retinoid pathway, distinct from that of retinoic acids.

B-Lymphocytes↗
PubMed1998

Pharmacology of irinotecan.

Irinotecan (CPT-11) is a semisynthetic derivative of camptothecin, an alkaloid extracted from the Chinese plant Camptotheca acuminata. It bears a bis-piperidine moiety and was selected for its water solubility and promising preclinical antitumor activity in in vitro and in vivo models. The target of drugs of the camptothecin family is DNA topoisomerase I, a nuclear enzyme involved in the relaxation of the DNA double helix required for replication and transcription activities. They stabilize the enzyme-DNA complex and prevent the religation of the single-strand breaks created by the enzyme, which are converted to double-strand breaks upon the collision with a replication fork during the S-phase. Resistance to irinotecan appears not to be mediated by P-glycoprotein, but by qualitative and/or quantitative alterations of its target, topoisomerase I, or by alterations occurring downstream of this interaction. As with all camptothecin derivatives, irinotecan contains a lactone ring that can be spontaneously and reversibly hydrolyzed to a carboxylate open ring form, which predominates at neutral and alkaline pH and is inactive on topoisomerase I-DNA complexes. Irinotecan is, in fact, much less active than its metabolite SN-38 and is generally considered as a prodrug of this compound. The carboxylesterase which carries out this conversion is preferentially active on the lactone form of irinotecan and directly generates the lactone form of SN-38, which may explain the superiority of irinotecan over SN-38 in vivo. Further metabolism of SN-38 to a beta-glucuronide conjugate is a major pathway of detoxification and plays an important role in determining irinotecan toxicity in the clinical setting. Other metabolic pathways of irinotecan involve oxidations occurring on the bis-piperidine rings, which are carried out by cytochrome P450. Irinotecan has shown an important activity in advanced and metastatic colorectal carcinoma and is now used for this indication in several countries, with two different recommended schedules: weekly administration of 125 mg/m(2) with a 2-week drug-free interval every 4 administrations or 3-weekly administration of 350 mg/m(2), a dose that can be increased to 500 mg/m(2) with the support of antidiarrhetics. Other possible indications of irinotecan include lung and cervix cancer, which are presently under investigation. The dose-limiting toxicity of irinotecan is mainly diarrhea, which occurs 7-10 days after treatment and can be life-threatening when associated with neutropenia, another frequent side effect. High-dose loperamide has shown good efficacy for treating this diarrhea and has allowed an increase in irinotecan doses tolerated by patients. The pharmacokinetics of irinotecan are characterized by a 2- or 3-compartment decay, with a terminal half-life of about 10 h, a total volume of distribution of 150 l/m(2) and a total plasma clearance of 15 l/h/m(2). SN-38 AUC is only a small fraction of that of irinotecan (2-4%) and SN-38 is eliminated from plasma with a half-life of about 12 h. SN-38 glucuronide is present in plasma at higher concentrations than SN-38 and is eliminated at the same rate. APC, produced by the action of cytochrome P450, isoenzyme 3A4, is present in plasma at concentrations close to those of irinotecan itself. Only a small fraction of irinotecan and its metabolites is eliminated in urine and a higher proportion in the bile, with an enterohepatic cycle of SN-38 glucuronide and SN-38. Significant relationships have been established between the AUCs of both irinotecan and SN-38 and hematological and intestinal toxicities, suggesting a potential use for monitoring of this drug.

Journal Article↗
PubMed1986

Glyceraldehyde-3-phosphate dehydrogenase is a nonhistone protein and a possible activator of transcription in neurons.

A single-stranded DNA-binding protein of Mr 35,000 (35K protein) was isolated from calf cerebral cortex by affinity chromatography on immobilized double-stranded and single-stranded DNA. Its localization in the nuclear compartment was demonstrated by immunohistochemistry. Previous studies had uncovered a homologous nonhistone chromosomal protein in the nuclei of rat cerebral cortex neurons, cerebellar neurons, oligodendrocytes, and liver cells. The rat protein accumulated in the nuclear compartment of neurons in exact temporal coincidence with the arrest of cell division and the initiation of terminal differentiation. Therefore, in the present work, the 35K protein was tested for an activating role in RNA transcription. During the course of this study we became aware that the 35K protein was identical to a glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). When authentic GAPDH from rabbit skeletal muscle was injected into Xenopus laevis oocytes, it greatly stimulated RNA polymerase II transcription, whereas the 35K protein from calf brain did not. This apparent discrepancy was partially resolved by the finding that rabbit muscle GAPDH could be fractionated into two components by affinity chromatography on single-stranded DNA cellulose. Only 5% of the applied protein was retained on the column and could be eluted with a shallow salt gradient identical to the one used for the isolation of the 35K protein. This single-stranded DNA-binding component of rabbit muscle GAPDH did not stimulate transcription. Apparently, the 35K protein from calf brain corresponded to this single-stranded DNA-binding subfraction, which explained its failure to activate transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Estimation of insulin secretion rates from C-peptide levels. Comparison of individual and standard kinetic parameters for C-peptide clearance.

Insulin secretion rates can be accurately estimated from plasma C-peptide levels with a two-compartment model for C-peptide distribution and degradation. In previous studies, the kinetic parameters of C-peptide clearance were derived in each subject from the decay curve observed after bolus intravenous injection of biosynthetic human C-peptide. To determine whether standard parameters for C-peptide clearance could be defined and used to calculate insulin secretion without obtaining a decay curve in each subject, we analyzed 200 decay curves of biosynthetic human C-peptide obtained in normal, obese, and non-insulin-dependent diabetes mellitus subjects studied in our laboratory. This analysis showed that the volume of distribution and kinetic parameters of C-peptide distribution and metabolism vary by less than 30% in a population highly heterogeneous in terms of age, sex, degree of obesity, and degree of glucose tolerance. The volume of distribution correlated with the degree of obesity as quantified by body surface area (BSA). This dependence of C-peptide distribution volume on BSA was more marked in men than in women. The long half-life was slightly longer in elderly subjects than in younger adults. When effects of BSA, sex, and age were taken into account, the parameters of C-peptide kinetics were very similar in normal, obese, and diabetic subjects. Based on these findings, a simple procedure to derive standard parameters for C-peptide clearance taking into account degree of obesity, sex, and age was defined. These standard parameters resulted in estimations of mean insulin secretion rates, which differed in each subject by only 10-12% from those obtained with individual parameters. The approach of using standard rather than individual parameters did not systematically underestimate or overestimate insulin secretion so that group values for the fasting secretion rate, the mean 24-h secretion rate, and the number and the amplitude of secretory pulses obtained with standard parameters differed by only 1-2% from the values obtained with individual parameters. Furthermore, the accuracy of measurements based on standard parameters was not different from that associated with replicate determinations of the parameters of C-peptide clearance in the same subject. We conclude that it is possible to estimate insulin secretion rates from plasma C-peptide levels with standard parameters for C-peptide clearance rather than individually derived parameters without significant loss of accuracy.

Adult↗
PubMed1990

Inhibition of tumour development in the partially resected, proliferating rat urinary bladder.

Autoradiographic studies have shown that the urothelium of the rat urinary bladder is capable to considerably proliferate in response to a partial cystectomy (one-third resection of the bladder) as is indicated by a 190-fold increase of the 3H-thymidine labelling index above normal levels 45 h postoperatively and an enormous increase of the compartment of proliferating cells (so-called growth fraction). The stimulated urothelial proliferation can be synchronized by multiple, fractionated doses of hydroxyurea (HU), resulting in a high degree of synchrony. Based on these findings, the partial cystectomy model seemed to be a useful tool to examine whether stimulated proliferation exerts a modifying effect on initiation of urothelial carcinogenesis. Following feeding N-butyl-N-(4-hydroxybutyl)-nitrosamine by gavage in 3 fractionated doses during most pronounced proliferation 30, 45 and 70 h postoperatively, the development of bladder tumors proved to be significantly dose- and time-related inhibited. Accordingly, N-methyl-N-nitrosourea (MNU)-induced tumour formation was considerably reduced following intravesicular instillation of the carcinogen at a single dose 45 h postoperatively, when stimulated DNA synthesis reached its peak. Experiments testing a possible cell cycle specific dependence of MNU-initiated tumor development in the partially resected bladder after synchronization of the stimulated proliferation by HU revealed an inhibition of urothelial carcinogenesis in particular, when the carcinogen was administered during the early DNA synthesis phase. The mechanisms underlying the observed inhibition of tumor development in the regeneration urinary bladder are unknown. It is assumed that an increased capacity of the proliferating urothelial cells to repair carcinogen-induced DNA-damage may play an important role.

Animals↗
PubMed2002

Minichromosome maintenance protein 2 expression in normal kidney and renal cell carcinomas: relationship to tumor dormancy and potential clinical utility.

PURPOSE: A major problem in the management of patients with renal cell carcinoma is predicting tumor behavior. In the search for more accurate markers of prognosis, tumor cell proliferation has been investigated. These studies have mostly used antibodies directed against Ki-67 or proliferating cell nuclear antigen and have given conflicting results, findings that are likely because of a combination of specificity and methodological differences. Minichromosome maintenance (Mcm) proteins are a series of closely related proteins that are components of the prereplicative complex. The Mcm proteins are essential for initiating eukaryotic DNA replication and serve as useful markers of proliferating cells. EXPERIMENTAL DESIGN: The aims of this study were to determine the frequency and pattern of Mcm2 expression by immunohistochemistry in normal kidney (n = 10) and renal tumors [n = 56; clear cell n = 36; chromophil (papillary) n = 7; oncocytoma n = 5; and transitional cell carcinoma n = 8], compare its sensitivity to the established proliferation marker Ki-67, examine for differences in tumors derived from stable and labile epithelial cell populations in the kidney, and assess the relationship of Mcm2 proliferation to clinicopathological characteristics of kidney tumors. In addition, to additionally investigate the issue of tumor dormancy we wished to assess the relationship between Mcm2 labeling index (LI) and the angiogenic factors angiopoietin-1 (Ang) and Ang-2. RESULTS: In normal tissues, Mcm2 nuclear labeling was identified in both glomeruli (LI median 0.35%; range 0-1.7) and renal tubules (LI median 0.3%; range 0.1-2.9%). In tumors Mcm2 labeling was predominantly at the periphery with LIs ranging from 0.2-91.5%, which was significantly greater than Ki-67 LI (0.2-40.5%; P < 0.001). Mcm2 LI was also significantly higher in tumors derived from a labile epithelium (transitional cell carcinomas) than a stable epithelium (renal cell carcinomas; P = 0.013). A significant association was also demonstrated between Mcm2 LI and tumor grade (P = 0.0006), and angiogenic phenotype (defined by Ang expression; P = 0.03) but not with patient age (P = 0.84), patient sex (P = 0.25), tumor size (P = 0.74), or stage (P = 0.33). Furthermore, although not significant, a survival analysis demonstrated that 100% of patients with a low Mcm2 LI survived compared with 84% of those with a high Mcm2 LI over the follow-up period (up to 53.2 months; P = 0.14). CONCLUSIONS: This is the first study examining Mcm2 protein in normal and tumor kidney samples, and the first to perform histological subgroup analysis. It shows that Mcm2 is a superior marker to Ki-67 in the assessment of cell cycle entry in histological archival material and that normal kidney has a subset of cells within the glomerular and tubular compartments that are in cycle. It demonstrates that the frequency of cells in cycle in tumors formed from stable or labile epithelial populations mirrors that in the nonneoplastic epithelium. This study additionally demonstrates that the number of cells in cycle in tumors is limited by the angiogenic phenotype and supports animal models that show angiogenesis determines the likelihood of tumor dormancy. Additional study to confirm the clinical utility of Mcm2 as a prognostic marker is now indicated.

Adult↗
PubMed1992

The liver as a stem cell and lineage system.

We propose that the liver is a stem cell and lineage system with many parallels to lineages in the bone marrow, gut, and epidermis, varying from them only in kinetics. All are organized with three compartments: a slow cycling stem cell compartment with cells expressing a fetal phenotype and responding slowly to injury; an amplification compartment with cells of intermediate phenotype rapidly proliferating in response to regenerative stimuli or acute injuries; and a terminal differentiation compartment in which cells increasingly differentiate and gradually lose their ability to divide. In all systems, both those with slow or rapid kinetics, the various compartments are positioned in a polarized organization, are associated with a gradient in the chemistry of the extracellular matrix, and show lineage-position-dependent growth responses, gene expression, pharmacological and toxicological responses, and reaction to viruses and radiation. In general, known oncogens selectively kill cells in the differentiation compartment inducing chronic regenerative responses of the cells in stem cell and/or amplification compartment. Tumors arise by subsequent transformation of the activated stem cells or early precursor cells. The evidence for a lineage model consists of the data implicating gradients in cell size, ploidy, growth potential, and antigenic and gene expression in the liver parenchyma along the sinusoidal plates. The traditional explanation for this heterogeneity is that it represents adaption of cells to a changing sinusoidal microenvironment dictated by the direction of blood flow. However, we review the extant data and suggest that it more readily supports a lineage model involving a maturation process beginning with stem cells and precursors in the periportal zone and ending with sensescing parenchyma near the central vein. Support for this theory is provided by the studies on phenotypic heterogeneity in liver, investigations into the embryology of the liver, and analyses of the responses of liver to chemical and viral oncogens that induce rapid proliferation of small cells with oval-shaped nuclei, "oval cells," now thought to be closely related to liver stem cells. The lineage model provides clarity and insights into many aspects of liver biology and disease including the limited proliferative ability of in vitro parenchymal cultures, liver regeneration, gene expression, viral infection, hepatocellular carcinogenesis, liver cell transplantation, and aging.

Aging↗
PubMed1998

Reflux of HTLV-I infected lymphocytes from the privileged compartment(s) to peripheral blood flow in patients with HTLV-I-associated myelopathy.

To understand the mechanisms involved in the pathogenesis of human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), three in vivo phenomena which have been observed in the peripheral blood of patients and differing from that in asymptomatic HTLV-I carriers must be taken into consideration: (a) the presence of increased HTLV-I viral load, (b) a higher immune responsiveness against HTLV-I antigens, and (c) biased nucleotide substitutions in the HTLV-I pX region which indicate a decreased selection pressure for viral amino acid changes. We now propose a hypothesis which focuses on the in vivo dynamics of HTLV-I infected lymphocyte migration and which incorporates these features. In addition, the hypothesis assumes the existence of a deviation in immune surveillance for HTLV-I in the central nervous system (CNS) in spite of the presence of frequent specific immune effectors. We suggest that in the active phase of HAM/TSP, accompanied with or following autoaggressive interactions between infected lymphocytes and immunocompetent cells in the CNS, there is a consequential reflux of the infected lymphocytes to the peripheral blood. The reflux of infected cells would be expected to provide peripheral blood with tissue-derived HTLV-I proviruses which have been indulged and propagated in an immune-privileged site. This process would result in and account for the observed increase in viral load and the substitution bias in HTLV-I sequences in the peripheral blood.

Deltaretrovirus Antibodies↗
PubMed1990

Clonal distribution of osteoprogenitor cells in cultured chick periostea: functional relationship to bone formation.

Folded explants of periosteum from embryonic chick calvaria form bone-like tissue when grown in the presence of ascorbic acid, organic phosphate, and dexamethasone. All osteoblast-like cells in these cultures arise de novo by differentiation of osteoprogenitor cells present in the periosteum. To study the spatial and functional relationships between bone formation and osteoprogenitor cells, cultures were continuously labeled with [3H]thymidine for periods of 1-5 days. Radioautographs of serial 2-microns plastic sections stained for alkaline phosphatase (AP) showed maximal labeling of 30% of fibroblastic (AP-negative) cells by 3 days while osteogenic cells (AP-positive) exhibited over 95% labeling by 5 days. No differential shifts in labeling indices, grain count histograms of fibroblastic and osteogenic cells or numbers of AP-positive cells were observed, indicating no significant recruitment of cells from the fibroblastic to the osteogenic compartment. Despite the continuous presence of [3H]thymidine, less than 35% of both osteoblasts and osteocytes were labeled at 5 days, indicating that only one-third of the osteoprogenitor cells had cycled prior to differentiation. Spatial clustering of [3H]thymidine-labeled cells was measured by computer-assisted morphometry and application of the Poisson distribution to assess contagion. Cluster size and number of labeled cells per cluster did not vary between 1-3 days, but the number of clusters increased 20-fold between Day 1 and Day 3. Clusters were predominantly AP-positive and located close to bone. Three-dimensional reconstruction from serial sections showed that clusters formed long, tubular arrays of osteogenic cells up to eight cells in length and located within 2-3 cell layers from the bone surface. Selective killing of S-phase cells with two pulse labels of high specific activity [3H]thymidine at 1 and 2 days of culture completely blocked bone formation. These data indicate that a very small population of cycling osteoprogenitor cells is essential for bone formation in vitro and give rise to relatively small numbers of clonally distributed progenitors with limited proliferative capacity. The progeny of these clusters undergo restricted migration and differentiate into osteoblasts.

Alkaline Phosphatase↗