Cellular responses to etoposide: cell death despite cell cycle arrest and repair of DNA damage

The topoisomerase IIα inhibitor etoposide is a ‘broad spectrum’ anticancer agent and a potent inducer of DNA double strand breaks. DNA damage response of mammalian cells usually involves cell cycle arrest and DNA repair or, if unsuccessful, cell death. We investigated these processes in the human colon cancer cell line HT-29 treated with three different etoposide regimens mimicking clinically relevant plasma concentrations of cancer patients. Each involved a period of drug-free incubation following etoposide exposure to imitate the decline of plasma levels between the cycles of chemotherapy. We found a massive induction of double strand breaks that were rapidly and nearly completely fixed long before the majority of cells underwent apoptosis or necrosis. An even greater percentage of cells lost clonogenicity. The occurrence of double strand breaks was accompanied by a decrease in the levels of Ku70, Ku86 and DNA-PK_cs as well as an increase in the level of Rad51 protein. Twenty-four hours after the first contact with etoposide we found a pronounced G₂/M arrest, regardless of the duration of drug exposure, the level of double strand breaks and the extent of their repair. During the subsequent drug-free incubation period, the loss of clonogenicity correlated well with the preceding G₂/M arrest as well as with the amount of cell death found several days after exposure. However, it correlated neither with early apoptosis or necrosis nor with any of the other investigated parameters. These results suggest that the G₂/M arrest is an important determinant in the cytostatic action of etoposide and that the removal of DNA double strand breaks is not sufficient to ensure cell survival.     

Entrapment Bias of Arthropods in Miocene Amber Revealed by Trapping Experiments in a Tropical Forest in Chiapas,Mexico

All entomological traps have a capturing bias, and amber, viewed as a trap, is no exception. Thus the fauna trapped in amber does not represent the total existing fauna of the former amber forest, rather the fauna living in and around the resin producing tree. In this paper we compare arthropods from a forest very similar to the reconstruction of the Miocene Mexican amber forest, and determine the bias of different trapping methods, including amber. We also show, using cluster analyses, measurements of the trapped arthropods, and guild distribution, that the amber trap is a complex entomological trap not comparable with a single artificial trap. At the order level, the most similar trap to amber is the sticky trap. However, in the case of Diptera, at the family level, the Malaise trap is also very similar to amber. Amber captured a higher diversity of arthropods than each of the artificial traps, based on our study of Mexican amber from the Middle Miocene, a time of climate optimum, where temperature and humidity were probably higher than in modern Central America. We conclude that the size bias is qualitatively independent of the kind of trap for non–extreme values. We suggest that frequent specimens in amber were not necessarily the most frequent arthropods in the former amber forest. Selected taxa with higher numbers of specimens appear in amber because of their ecology and behavior, usually closely related with a tree–inhabiting life. Finally, changes of diversity from the Middle Miocene to Recent time in Central and South America can be analyzed by comparing the rich amber faunas from Mexico and the Dominican Republic with the fauna trapped using sticky and Malaise traps in Central America.     

Growth and biochemical characteristics of a detachment variant of CHO cells.

A variant subline of Chinese hamster cells (line CHO) was isolated that had increased resistance to detachment from the substratum. Comparisons between parental and variant cells of the complex carbohydrates liberated during trypsin detachment showed that the variant cells synthesized little or no hyaluronic acid. These cells also had reduced amounts of other complex carbohydrates in the cell periphery. However, parental and variant cells did not differ in morphology, growth control, or cyclic AMP concentration. Profound changes in the physical nature of the cell periphery, in themselves, evidently are insufficient to cause changes in many aspects of cell behavior.     

Oxygen consumption following exercise of moderate intensity and duration.

To study the effects of exercise intensity and duration on excess postexercise oxygen consumption (EPOC), 8 men [age = 27.6 (SD 3.8) years, VO2max = 46.1 (SD 8.5) ml min-1 kg-1] performed four randomly assigned cycle-ergometer tests (20 min at 60% VO2max, 40 min at 60% VO2max, 20 min at 70% VO2max, and 40 min at 70% VO2max). O2 uptake, heart rate and rectal temperature were measured before, during, and for 1 h following the exercise tests. Blood for plasma lactate measurements was obtained via cannulae before, and at selected times, during and following exercise. VO2 rapidly declined to preexercise levels following each of the four testing sessions, and there were no differences in EPOC between the sessions. Blood lactate and rectal temperature increased (P < 0.05) with exercise, but had returned to preexercise levels by 40 min of recovery. The results indicate that VO2 returned to resting levels within 40 min after the end of exercise, regardless of the intensity (60% and 70% VO2max) or duration (20 min and 40 min) of the exercise, in men with a moderate aerobic fitness level.     

Age-Related Modulation of the Effects of Obesity on Gene Expression Profiles of Mouse Bone Marrow and Epididymal Adipocytes

This study aimed to characterize and compare the effects of obesity on gene expression profiles in two distinct adipose depots, epididymal and bone marrow, at two different ages in mice. Alterations in gene expression were analyzed in adipocytes isolated from diet-induced obese (DIO) C57BL/6J male mice at 6 and 14 months of age and from leptin deficient mice (ob/ob) at 6 months of age using microarrays. DIO affected gene expression in both depots at 6 and 14 months, but more genes were altered in epididymal than bone marrow adipocytes at each age and younger mice displayed more changes than older animals. In epididymal adipocytes a total of 2789 (9.6%) genes were differentially expressed at 6-months with DIO, whereas 952 (3.3%) were affected at 14-months. In bone marrow adipocytes, 347 (1.2%) genes were differentially expressed at 6-months with DIO, whereas only 189 (0.66%) were changed at 14-months. 133 genes were altered by DIO in both fat depots at 6-months, and 37 genes at 14-months. Only four genes were altered in both depots at both ages with DIO. Bone marrow adipocytes are less responsive to DIO than epididymal adipocytes and the response of both depots to DIO declines with age. This loss of responsiveness with age is likely due to age-associated changes in expression of genes related to adipogenesis, inflammation and mitochondrial function that are similar to and obscure the changes commonly associated with DIO. Patterns of gene expression were generally similar in epididymal adipocytes from ob/ob and DIO mice; however, several genes were differentially expressed in bone marrow adipocytes from ob/ob and DIO mice, perhaps reflecting the importance of leptin signaling for bone metabolism. In conclusion, obesity affects age-associated alterations in gene expression in both epididymal and bone marrow adipocytes regardless of diet or genetic background.     

Revisiting recent etiological theories of functions

Arguably, the most widely endorsed account of normative functions in philosophy of biology is an etiological theory that holds that the function of current traits is fixed by the past selection history of other traits of that type. The earlier formulations of this “selected-effects” theory had trouble accommodating vestigial traits. In order to remedy these difficulties, the influential recent selection or modern history selected-effects theory was introduced. This paper expands upon and strengthens the argument that this theory has trouble stemming from recent “no variation” cases. In addition, several influential arguments for the necessity of including a selection requirement in a theory of normative biological functions are contested. It is suggested that accounting for biological functions in certain areas of biology (such as physiology and the neurosciences) does not require adverting to selection.     

Unusual Spectral Properties of Bacteriophytochrome Agp2 Result from a Deprotonation of the Chromophore in the Red-absorbing Form Pr

Phytochromes are widely distributed photoreceptors with a bilin chromophore that undergo a typical reversible photoconversion between the two spectrally different forms, Pr and Pfr. The phytochrome Agp2 from Agrobacterium tumefaciens belongs to the group of bathy phytochromes that have a Pfr ground state as a result of the Pr to Pfr dark conversion. Agp2 has untypical spectral properties in the Pr form reminiscent of a deprotonated chromophore as confirmed by resonance Raman spectroscopy. UV/visible absorption spectroscopy showed that the pK_a is >11 in the Pfr form and ∼7.6 in the Pr form. Unlike other phytochromes, photoconversion thus results in a pK_a shift of more than 3 units. The Pr/Pfr ratio after saturating irradiation with monochromatic light is strongly pH-dependent. This is partially due to a back-reaction of the deprotonated Pr chromophore at pH 9 after photoexcitation as found by flash photolysis. The chromophore protonation and dark conversion were affected by domain swapping and site-directed mutagenesis. A replacement of the PAS or GAF domain by the respective domain of the prototypical phytochrome Agp1 resulted in a protonated Pr chromophore; the GAF domain replacement afforded an inversion of the dark conversion. A reversion was also obtained with the triple mutant N12S/Q190L/H248Q, whereas each single point mutant is characterized by decelerated Pr to Pfr dark conversion.