Examination of synthetic retinoid -induced apoptosis in cutaneous squamous cell carcinomas: Mechanisms and implications for chemoprevention and chemotherapy with retinoids

Non-melanoma skin cancers, including basal cell carcinoma and squamous cell carcinoma (SCC), are the most common neoplasms in the United States with a lifetime risk nearly equal to all other types of cancer combined. Retinoids are naturally occurring and synthetic analogues of vitamin A that bind to nuclear retinoid receptors and modulate gene expression as a means of regulating cell proliferation and differentiation. Retinoids have been employed for many years in the treatment of various cutaneous lesions and for cancer chemoprevention and therapy. The primary drawback limiting the use of retinoids is their toxicity, which is also associated with receptor-gene interactions. In this study, the effects of the synthetic retinoids N-(4-hydroxyphenyl)retinamide (4HPR) and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) were examined in cutaneous keratinocytes. Four human cutaneous SCC cell lines were examined along with normal human epidermal keratinocyte (NHEK) cells from two donors. Sensitivity to 4HPR or CD437 alone or in combination with other agents was determined via growth inhibition, cell cycle distributions, or apoptosis induction. Both synthetic retinoids were able to promote apoptosis in SCC cells more effectively than the natural retinoid all-trans retinoic acid. Apoptosis could not be inhibited by nuclear retinoic acid receptor antagonists. In NHEK cells, 4HPR induced apoptosis while CD437 promoted G1 arrest. 4HPR acted as a prooxidant by generating reactive oxygen species (ROS) in SCC and NHEK cells. 4HPR-induced apoptosis in SCC cells could be inhibited or potentiated by manipulating cellular defenses against oxidative stress, indicating an essential role for ROS in 4HPR-induced apoptosis. CD437 promoted apoptosis in SCC cells in S and G2/M phases of the cell cycle within two hours of treatment, and this rapid induction could not be blocked with cycloheximide. This study shows: (1) 4HPR- and CD437-induced apoptosis do not directly involve a traditional retinoid pathway; (2) 4HPR can act as a prooxidant as a means of promoting apoptosis; (3) CD437 induces apoptosis in SCC cells independent of protein synthesis and is potentially less toxic to NHEK cells; and (4) 4HPR and CD437 operate under different mechanisms with respect to apoptosis induction and this may potentially enhance their therapeutic index in vivo. ^

Análisis de la marcha: evaluación de un exoesqueleto aplicado a la marcha asistida

La marcha humana es el mecanismo de locomoción por el cual el cuerpo humano se traslada en línea recta gracias a una serie de movimientos coordinados de la pelvis y de las articulaciones del miembro inferior. Frecuentemente se encuentra influenciada por factores biomecánicos, anatómicos o patologías del sistema neuromusculoesquelético que modifican la forma de caminar de cada individuo. La lesión de médula espinal es una de las patologías que afectan el desarrollo normal de los patrones de la marcha por alteración de la movilidad, de la sensibilidad o del sistema nervioso autónomo. Aunque la lesión medular afecta otras funciones, además de la pérdida de función motora y sensorial, la recuperación de la capacidad de caminar es la mayor prioridad identificada por los pacientes durante la rehabilitación. Por ello, el desarrollo de dispositivos que faciliten la rehabilitación o compensación de la marcha es uno de los principales objetivos de diferentes grupos de investigación y empresas. En el contexto del proyecto Hybrid Technological Platform for Rehabilitation, Functional Compensation and Training of Gait in Spinal Cord Injury Patients se ha desarrollado un dispositivo que combina una órtesis activa (exoesqueleto) y un andador motorizado. Este sistema, como otros dispositivos, tiene el movimiento humano como estándar de referencia, no obstante no se evalúa de manera habitual, cómo es el patrón de la marcha reproducido y su similitud o diferencias con la marcha humana, o las modificaciones o adaptaciones en la interacción con el cuerpo del paciente. El presente estudio trata de examinar las características de la marcha normal en diversos grupos de población, y las diferencias con el patrón de marcha lenta. Finalmente, se pretende evaluar qué modificaciones y adaptaciones sufre el patrón de marcha lenta teórico al ser reproducido por el exoesqueleto. La presente investigación consiste en un estudio cuantitativo transversal desarrollado en dos etapas: estudio 1 y estudio 2. En el estudio 1 se analizó el patrón de la marcha a velocidad libremente seleccionada (normal) y el patrón de la marcha a velocidad lenta (0.25m/s) en 62 sujetos distribuidos en grupos considerando el sexo y los percentiles 25, 50 y 75 de estatura de la población española. Durante el estudio 2 se analizó el patrón de la marcha lenta reproducido por el dispositivo Hybrid a diferentes porcentajes de peso corporal (30%, 50% y 70%) en diez sujetos seleccionados aleatoriamente de la muestra del estudio 1. En ambos estudios se obtuvieron variables espacio-temporales y cinemáticas mediante un sistema de captura de movimiento con 6 cámaras distribuidas a lo largo de un pasillo de marcha. Se calcularon las medias, las desviaciones estándar y el 95% de intervalo de confianza, y el nivel alfa de significación se estableció en α=0.05 para todas las pruebas estadísticas. Las principales diferencias en el patrón normal de la marcha se encontraron en los parámetros cinemáticos de hombres y mujeres, aunque también se presentaron diferencias entre los grupos en función de la estatura. Las mujeres mostraron mayor flexión de cadera y rodilla, y mayor extensión de tobillo que los hombres durante el ciclo normal, aunque la basculación lateral de la pelvis, mayor en las mujeres, y el desplazamiento lateral del centro de gravedad, mayor en los hombres, fueron los parámetros identificados como principales discriminantes entre sexos. La disminución de la velocidad de la marcha mostró similares adaptaciones y modificaciones en hombres y en mujeres, presentándose un aumento de la fase de apoyo y una disminución de la fase de oscilación, un retraso de los máximos y mínimos de flexoextensión de cadera, rodilla y tobillo, y una disminución del rango articular en las tres articulaciones. Asimismo, la basculación lateral de la pelvis y el movimiento vertical del centro de gravedad disminuyeron, mientras que el movimiento lateral del centro de gravedad y el ancho de paso aumentaron. Durante la evaluación del patrón de la marcha reproducido por el exoesqueleto se observó que las tres articulaciones del miembro inferior disminuían el rango de movimiento por la falta de fuerza de los motores para contrarrestar el peso corporal, incluso con un 70% de descarga de peso. Además, la transferencia de peso se encontró limitada por la falta de movimiento de la pelvis en el plano frontal y se sustituyó por un aumento de la inclinación del tronco y, por tanto, del movimiento lateral del centro de gravedad. Este hecho, junto al aumento del desplazamiento vertical del centro de gravedad, hizo del patrón de la marcha reproducido por el exoesqueleto un movimiento poco eficiente. En conclusión, se establecen patrones de marcha normal diferenciados por sexos, siendo la basculación lateral de la pelvis y el movimiento lateral del centro de gravedad los parámetros discriminantes más característicos entre sexos. Comparando la marcha a velocidad libremente seleccionada y la velocidad lenta, se concluye que ambos sexos utilizan estrategias similares para adaptar el patrón de la marcha a una velocidad lenta y se mantienen las características diferenciadoras entre hombres y mujeres. En relación a la evaluación del dispositivo Hybrid, se deduce que la falta de movimiento lateral de la pelvis condiciona la transferencia de peso y el aumento del rango de movimiento del centro de gravedad y, en consecuencia, tiene como resultado un patrón de la marcha poco eficiente. Este patrón no resultaría indicado para los procesos de rehabilitación o recuperación de la marcha, aunque podría considerarse adecuado para la compensación funcional de la bipedestación y la locomoción. ABSTRACT The human walking is a means of moving body forward using a repetitious and coordinated sequence of pelvis and lower limb motions. It is frequently influenced by biomechanical and anatomical factors or by musculoskeletal pathologies which modify the way of walking. The spinal injury is one of those pathologies which affect the normal pattern of walking, due to the alteration of the mobility, the sensory or the autonomic nervous system. Although the spinal injury affects many other body functions, apart from the motor and sensory ones, the main priority for patients is to recover the ability of walking. Consequently, the main objective of many research groups and private companies is the development of rehabilitation and compensation devices for walking. In this context, the Hybrid Technological Platform for Rehabilitation, Functional Compensation and Training of Gait in Spinal Cord Injury Patients project has developed a device which integrates an exoskeleton and a motorized smart walker. This system, as other similar devices, has the human movement as standard reference. Nevertheless, these devices are not usually evaluated on the way they reproduce the normal human pattern or on the modifications and in the interactions with the patient’s body. The aim of the present study is to examine the normal walking characteristics, to analyze the differences between self-selected and low speed walking patterns, and to evaluate the modifications and adaptations of walking pattern when it is reproduced by the exoskeleton. The present research is a quantitative cross-sectional study carried out in two phases: study 1 and study 2. During the study 1, the self-selected and the low speed (0.25m/s) walking patterns were analyzed in sixty-two people distributed in groups, according to sex and 25th, 50th and 75th percentiles of height for Spanish population. The study 2 analyzed the low speed walking pattern reproduced by the Hybrid system in three conditions: 30%, 50% and 70% of body weight support. To do this, ten subjects were randomly selected and analyzed from the people of study 1. An optoelectronic system with six cameras was used to obtain spatial, temporal and kinematic parameters in both studies. Means, standard deviations and 95% confidence intervals of the study were calculated. The alpha level of significance was set at α=0.05 for all statistical tests. The main differences in normal gait pattern were found in kinematic parameters between men and women. The hip and the knee were more flexed and the ankle plantar flexion was higher in women than in men during normal gait cycle. Although the greater pelvic obliquity of women and the higher lateral movement of center of gravity of men were the most relevant discriminators between male and female gait patterns. Comparing self-selected and low speed walking patterns, both sexes showed similar adaptations and modifications. At low speed walking, men and women increased the stance phase ratio and decreased the swing phase ratio. The maximum and minimum peak flexion of hip, knee and ankle appeared after and the range of motion of them decreased during low speed walking. Furthermore, the pelvic obliquity and the vertical movement of the center of gravity decreased, whereas the lateral movement of center of gravity and step width increased. Evaluating the gait pattern reproduced by the exoskeleton, a decrease of lower limb range of motion was observed. This was probably due to the lack of strength of the engines, which were not able to control the body weight, even with the 70% supported. Moreover, the weight transfer from one limb to the contralateral side was restricted due to the lack of pelvis obliquity. This movement deficiency was replaced by the lateral torso sway and, consequently, the increase of lateral movement of the center of gravity. This fact, as well as the increase of the vertical displacement of the center of gravity, made inefficient the gait pattern reproduced by the exoskeleton. In conclusion, different gait patterns of both sexes have been determined, being pelvis obliquity and lateral movement of center of gravity the most relevant discriminators between male and female gait patterns. Comparing self-selected and low speed walking patterns, it was concluded that both sexes use similar strategies for adapting the gait pattern to a low speed, and therefore, the differentiating characteristics of normal gait are maintained. Regarding the Hybrid system evaluation, it was determined that the gait pattern reproduced by the exoskeleton is inefficient. This was due to the lack of pelvis obliquity and the increase of the center of gravity displacement. Consequently, whereas the walking pattern reproduced by the exoskeleton would not be appropriated for the rehabilitation process, it could be considered suitable for functional compensation of walking and standing.

Activity and nature of p21WAF1 complexes during the cell cycle

Elevated levels of the p21WAF1 (p21) cyclin-dependent kinase inhibitor induce growth arrest. We have characterized a panel of monoclonal antibodies against human p21 in an effort to understand the dynamic regulatory interactions between this and other cellular proteins during the cell cycle. The use of these reagents has allowed us to address several important, yet unresolved, issues concerning the biological activity of p21, including the potential kinase activity of complexes that associate with this cyclin-dependent kinase inhibitor. We have found that the kinase activity of cyclin A/Cdk2 associated with p21 is significantly lower than that of cyclin A/Cdk2 free of p21, suggesting that p21 abolishes its activity in vivo, and the use of multiple antibodies has enabled us to begin the study of the molecular architecture of p21 complexes in vivo. In addition, we found that human fibroblasts released from a quiescent state display abundant amounts of p21 devoid of associated proteins (“free” p21), the levels of which decrease as cells approach S phase. Cyclin A levels increase as the amount of monomeric p21 decreases, resulting in an excess of cyclin A/Cdk2 complexes that are not bound to, or inactivated by, p21. Our data strengthen the notion that the G1-to-S phase transition in human fibroblasts occurs when the concentration of cyclin A/Cdk2 surpasses that of p21.

Growth of Toxoplasma gondii is inhibited by aryloxyphenoxypropionate herbicides targeting acetyl-CoA carboxylase

Aryloxyphenoxypropionates, inhibitors of the plastid acetyl-CoA carboxylase (ACC) of grasses, also inhibit Toxoplasma gondii ACC. Clodinafop, the most effective of the herbicides tested, inhibits growth of T. gondii in human fibroblasts by 70% at 10 μM in 2 days and effectively eliminates the parasite in 2–4 days at 10–100 μM. Clodinafop is not toxic to the host cell even at much higher concentrations. Parasite growth inhibition by different herbicides is correlated with their ability to inhibit ACC enzyme activity, suggesting that ACC is a target for these agents. Fragments of genes encoding the biotin carboxylase domain of multidomain ACCs of T. gondii, Plasmodium falciparum, Plasmodium knowlesi, and Cryptosporidium parvum were sequenced. One T. gondii ACC (ACC1) amino acid sequence clusters with P. falciparum ACC, P. knowlesi ACC, and the putative Cyclotella cryptica chloroplast ACC. Another sequence (ACC2) clusters with that of C. parvum ACC, probably the cytosolic form.

Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization

The immortalization of human cells is a critical step during tumorigenesis. In vitro, normal human somatic cells must overcome two proliferative blockades, senescence and crisis, to become immortal. Transformation with viral oncogenes extends the life span of human cells beyond senescence. Such transformed cells eventually succumb to crisis, a period of widespread cellular death that has been proposed to be the result of telomeric shortening. We now show that ectopic expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) and subsequent activation of telomerase can allow postsenescent cells to proliferate beyond crisis, the last known proliferative blockade to cellular immortality. Moreover, we demonstrate that alteration of the carboxyl terminus of human telomerase reverse transcriptase does not affect telomerase enzymatic activity but impedes the ability of this enzyme to maintain telomeres. Telomerase-positive cells expressing this mutant enzyme fail to undergo immortalization, further tightening the connection between telomere maintenance and immortalization.

Katanin Is Responsible for the M-Phase Microtubule-severing Activity in Xenopus Eggs

Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role in cell cycle-dependent changes in microtubule dynamics and organization. However, the isolation of three different microtubule-severing proteins, p56, EF1α, and katanin, has only confused the issue because none of these proteins is directly activated by cyclin B/cdc2. Here we use immunodepletion with antibodies specific for a vertebrate katanin homologue to demonstrate that katanin is responsible for the majority of M-phase severing activity in Xenopus eggs. This result suggests that katanin is responsible for changes in microtubules occurring at mitosis. Immunofluorescence analysis demonstrated that katanin is concentrated at a microtubule-dependent structure at mitotic spindle poles in Xenopus A6 cells and in human fibroblasts, suggesting a specific role in microtubule disassembly at spindle poles. Surprisingly, katanin was also found in adult mouse brain, indicating that katanin may have other functions distinct from its mitotic role.

c-Abl is required for development and optimal cell proliferation in the context of p53 deficiency

The c-Abl tyrosine kinase and the p53 tumor suppressor protein interact functionally and biochemically in cellular genotoxic stress response pathways and are implicated as downstream mediators of ATM (ataxia-telangiectasia mutated). This fact led us to study genetic interactions in vivo between c-Abl and p53 by examining the phenotype of mice and cells deficient in both proteins. c-Abl-null mice show high neonatal mortality and decreased B lymphocytes, whereas p53-null mice are prone to tumor development. Surprisingly, mice doubly deficient in both c-Abl and p53 are not viable, suggesting that c-Abl and p53 together contribute to an essential function required for normal development. Fibroblasts lacking both c-Abl and p53 were similar to fibroblasts deficient in p53 alone, showing loss of the G1/S cell-cycle checkpoint and similar clonogenic survival after ionizing radiation. Fibroblasts deficient in both c-Abl and p53 show reduced growth in culture, as manifested by reduction in the rate of proliferation, saturation density, and colony formation, compared with fibroblasts lacking p53 alone. This defect could be restored by reconstitution of c-Abl expression. Taken together, these results indicate that the ATM phenotype cannot be explained solely by loss of c-Abl and p53 and that c-Abl contributes to enhanced proliferation of p53-deficient cells. Inhibition of c-Abl function may be a therapeutic strategy to target p53-deficient cells selectively.

Protective immunity to HIV-1 in SCID/beige mice reconstituted with peripheral blood lymphocytes of exposed but uninfected individuals

Immunodeficiency typically appears many years after initial HIV infection. This long, essentially asymptomatic period contributes to the transmission of HIV in human populations. In rare instances, clearance of HIV-1 infection has been observed, particularly in infants. There are also reports of individuals who have been frequently exposed to HIV-1 but remain seronegative for the virus, and it has been hypothesized that these individuals are resistant to infection by HIV-1. However, little is known about the mechanism of immune clearance or protection against HIV-1 in these high-risk individuals because it is difficult to directly demonstrate in vivo protective immunity. Although most of these high-risk individuals show an HIV-1-specific cell-mediated immune response using in vitro assays, their peripheral blood lymphocytes (PBLs) are still susceptible to HIV infection in tissue culture. To study this further in vivo, we have established a humanized SCID mouse infection model whereby T-, B-, and natural killer-cell defective SCID/beige mice that have been reconstituted with normal human PBLs can be infected with HIV-1. When the SCID/beige mice were reconstituted with PBLs from two different multiply exposed HIV-1 seronegative individuals, the mice showed resistance to infection by two strains of HIV-1 (macrophage tropic and T cell tropic), although the same PBLs were easily infected in vitro. Mice reconstituted with PBLs from non-HIV-exposed controls were readily infected. When the same reconstituted mice were depleted of human CD8 T cells, however, they became susceptible to HIV-1 infection, indicating that the in vivo protection required CD8 T cells. This provides clear experimental evidence that some multiply exposed, HIV-1-negative individuals have in vivo protective immunity that is CD8 T cell-dependent. Understanding the mechanism of such protective immunity is critical to the design and testing of effective prophylactic vaccines and immunotherapeutic regimens.

Infusion of α-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). This enzymatic defect results in the accumulation of the glycosphingolipid globotriaosylceramide (Gb3; also referred to as ceramidetrihexoside) throughout the body. To investigate the effects of purified α-gal A, 10 patients with Fabry disease received a single i.v. infusion of one of five escalating dose levels of the enzyme. The objectives of this study were: (i) to evaluate the safety of administered α-gal A, (ii) to assess the pharmacokinetics of i.v.-administered α-gal A in plasma and liver, and (iii) to determine the effect of this replacement enzyme on hepatic, urine sediment and plasma concentrations of Gb3. α-Gal A infusions were well tolerated in all patients. Immunohistochemical staining of liver tissue approximately 2 days after enzyme infusion identified α-gal A in several cell types, including sinusoidal endothelial cells, Kupffer cells, and hepatocytes, suggesting diffuse uptake via the mannose 6-phosphate receptor. The tissue half-life in the liver was greater than 24 hr. After the single dose of α-gal A, nine of the 10 patients had significantly reduced Gb3 levels both in the liver and shed renal tubular epithelial cells in the urine sediment. These data demonstrate that single infusions of α-gal A prepared from transfected human fibroblasts are both safe and biochemically active in patients with Fabry disease. The degree of substrate reduction seen in the study is potentially clinically significant in view of the fact that Gb3 burden in Fabry patients increases gradually over decades. Taken together, these results suggest that enzyme replacement is likely to be an effective therapy for patients with this metabolic disorder.

Retinoic acid induces sodium/iodide symporter gene expression and radioiodide uptake in the MCF-7 breast cancer cell line

The sodium/iodide symporter (NIS) stimulates iodide uptake in normal lactating breast, but is not known to be active in nonlactating breast or breast cancer. We studied NIS gene regulation and iodide uptake in MCF-7 cells, an estrogen receptor (ER)-positive human breast cancer cell line. All-trans retinoic acid (tRA) treatment stimulated iodide uptake in a time- and dose-dependent fashion up to ≈9.4-fold above baseline. Stimulation with selective retinoid compounds indicated that the induction of iodide uptake was mediated by retinoic acid receptor. Treatment with tRA markedly stimulated NIS mRNA and immunoreactive protein (≈68 kDa). tRA stimulated NIS gene transcription ≈4-fold, as shown by nuclear run-on assay. No induction of iodide uptake was observed with RA treatment of an ER-negative human breast cancer cell line, MDA-MB 231, or a normal human breast cell line, MCF-12A. The iodide efflux rate of tRA-treated MCF-7 cells was slow (t1/2 = 24 min), compared with that in FRTL-5 thyroid cells (t1/2 = 3.9 min), favoring iodide retention in MCF-7 cells. An in vitro clonogenic assay demonstrated selective cytotoxicity with 131I after tRA stimulation of MCF-7 cells. tRA up-regulates NIS gene expression and iodide uptake in an ER-positive breast cancer cell line. Stimulation of radioiodide uptake after systemic retinoid treatment may be useful for diagnosis and treatment of some differentiated breast cancers.

Orbitofrontal cortex: A key prefrontal region for encoding information

Little is known about the specific functional contribution of the human orbitofrontal cortex with regard to memory processing, although there is strong evidence from lesion studies in monkeys that it may play an important role. The present investigation measured changes in regional cerebral blood flow with positron emission tomography in normal human subjects who were instructed to commit to memory abstract visual patterns. The results indicated that the rostral orbitofrontal region (area 11), which is primarily linked with the anterior medial temporal limbic region and lateral prefrontal cortical areas, is involved in the process of encoding of new information.

UV-induced inhibition of transcription involves repression of transcription initiation and phosphorylation of RNA polymerase II

Cells from patients with Cockayne syndrome (CS) are hypersensitive to DNA-damaging agents and are unable to restore damage-inhibited RNA synthesis. On the basis of repair kinetics of different types of lesions in transcriptionally active genes, we hypothesized previously that impaired transcription in CS cells is a consequence of defective transcription initiation after DNA damage induction. Here, we investigated the effect of UV irradiation on transcription by using an in vitro transcription system that allowed uncoupling of initiation from elongation events. Nuclear extracts prepared from UV-irradiated or mock-treated normal human and CS cells were assayed for transcription activity on an undamaged β-globin template. Transcription activity in nuclear extracts closely mimicked kinetics of transcription in intact cells: extracts from normal cells prepared 1 h after UV exposure showed a strongly reduced activity, whereas transcription activity was fully restored in extracts prepared 6 h after treatment. Extracts from CS cells exhibited reduced transcription activity at any time after UV exposure. Reduced transcription activity in extracts coincided with a strong reduction of RNA polymerase II (RNAPII) containing hypophosphorylated C-terminal domain, the form of RNAPII known to be recruited to the initiation complex. These results suggest that inhibition of transcription after UV irradiation is at least partially caused by repression of transcription initiation and not solely by blocked elongation at sites of lesions. Generation of hypophosphorylated RNAPII after DNA damage appears to play a crucial role in restoration of transcription. CS proteins may be required for this process in a yet unknown way.

Platelet-derived growth factor activates mitogen-activated protein kinase in isolated caveolae

The ability of a peptide hormone to affect many different intracellular targets is thought to be possible because of the modular organization of signal transducing molecules in the cell. Evidence for the presence of signaling modules in metazoan cells, however, is incomplete. Herein we show, with morphology and cell fractionation, that all the components of a mitogen-activated protein kinase pathway are concentrated in caveolae of unstimulated human fibroblasts. Addition of platelet-derived growth factor to either the intact cell or caveolae isolated from these cells stimulates tyrosine phosphorylation and activates mitogen-activated protein kinases in caveolae. The molecular machinery for kinase activation, therefore, is preorganized at the cell surface of quiescent cells.

Telomerase activity: A biomarker of cell proliferation, not malignant transformation

Telomerase activity is readily detected in most cancer biopsies, but not in premalignant lesions or in normal tissue samples with a few exceptions that include germ cells and hemopoietic stem cells. Telomerase activity may, therefore, be a useful biomarker for diagnosis of malignancies and a target for inactivation in chemotherapy or gene therapy. These observations have led to the hypothesis that activation of telomerase may be an important step in tumorigenesis. To test this hypothesis, we studied telomerase activity in isogeneic samples of uncultured and cultured specimens of normal human uroepithelial cells (HUCs) and in uncultured and cultured biopsies of superficial and myoinvasive transitional cell carcinoma (TCC) of the bladder. Our results demonstrated that four of four TCC biopsies, representing both superficial and myoinvasive TCCs, were positive for telomerase activity, but all samples of uncultured HUC were telomerase negative. However, when the same normal HUC samples were established as proliferating cultures in vitro, telomerase activity was readily detected but usually at lower levels than in TCCs. Consistent with the above observation of the telomerase activity in HUCs, telomeres did not shorten during the HUC in vitro lifespan. Demonstration of telomerase in proliferating human epithelial cells in vitro was not restricted to HUCs, because it was also present in prostate and mammary cell cultures. Notably, telomerase activity was relatively low or undetectable in nonproliferating HUC cultures. These data do not support a model in which telomerase is inactive in normal cells and activated during tumorigenic transformation. Rather, these data support a model in which the detection of telomerase in TCC biopsies, but not uncultured HUC samples, reflects differences in proliferation between tumor and normal cells in vivo.

Genetic evidence for the involvement of DNA ligase IV in the DNA-PK-dependent pathway of non-homologous end joining in mammalian cells

Cells of vertebrates remove DNA double-strand breaks (DSBs) from their genome predominantly utilizing a fast, DNA-PKcs-dependent form of non-homologous end joining (D-NHEJ). Mutants with inactive DNA-PKcs remove the majority of DNA DSBs utilizing a slow, DNA-PKcs-independent pathway that does not utilize genes of the RAD52 epistasis group, is error-prone and can therefore be classified as a form of NHEJ (termed basic or B-NHEJ). We studied the role of DNA ligase IV in these pathways of NHEJ. Although biochemical studies show physical and functional interactions between the DNA-PKcs/Ku and the DNA ligase IV/Xrcc4 complexes suggesting operation within the same pathway, genetic evidence to support this notion is lacking in mammalian cells. Primary human fibroblasts (180BR) with an inactivating mutation in DNA ligase IV, rejoined DNA DSBs predominantly with slow kinetics similar to those observed in cells deficient in DNA-PKcs, or in wild-type cells treated with wortmannin to inactivate DNA-PK. Treatment of 180BR cells with wortmannin had only a small effect on DNA DSB rejoining and no effect on cell radiosensitivity to killing although it sensitized control cells to 180BR levels. This is consistent with DNA ligase IV functioning as a component of the D-NHEJ, and demonstrates the unperturbed operation of the DNA-PKcs-independent pathway (B-NHEJ) at significantly reduced levels of DNA ligase IV. In vitro, extracts of 180BR cells supported end joining of restriction endonuclease-digested plasmid to the same degree as extracts of control cells when tested at 10 mM Mg2+. At 0.5 mM Mg2+, where only DNA ligase IV is expected to retain activity, low levels of end joining (∼10% of 10 mM) were seen in the control but there was no detectable activity in 180BR cells. Antibodies raised against DNA ligase IV did not measurably inhibit end joining at 10 mM Mg2+ in either cell line. Thus, in contrast to the situation in vivo, end joining in vitro is dominated by pathways with properties similar to B-NHEJ that do not display a strong dependence on DNA ligase IV, with D-NHEJ retaining only a limited contribution. The implications of these observations to studies of NHEJ in vivo and in vitro are discussed.