The KRAS-regulated kinome identifies WEE1 and ERK coinhibition as a potential therapeutic strategy in KRAS-mutant pancreatic cancer

Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines and then applied multiplexed inhibitor bead/MS to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transformation and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel of cell lines. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGF-β1, ILK), and pharmacological inhibition of one of these upregulated kinases, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacological inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death compared with WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.     

Adaptation to Ephemeral Habitat May Overcome Natural Barriers and Severe Habitat Fragmentation in a Fire-Dependent Species,the Bachman's Sparrow (Peucaea aestivalis)

Bachman''s Sparrow (Peucaea aestivalis) is a fire-dependent species that has undergone range-wide population declines in recent decades. We examined genetic diversity in Bachman''s Sparrows to determine whether natural barriers have led to distinct population units and to assess the effect of anthropogenic habitat loss and fragmentation. Genetic diversity was examined across the geographic range by genotyping 226 individuals at 18 microsatellite loci and sequencing 48 individuals at mitochondrial and nuclear genes. Multiple analyses consistently demonstrated little genetic structure and high levels of genetic variation, suggesting that populations are panmictic. Based on these genetic data, separate management units/subspecies designations or translocations to promote gene flow among fragmented populations do not appear to be necessary. Panmixia in Bachman''s Sparrow may be a consequence of an historical range expansion and retraction. Alternatively, high vagility in Bachman''s Sparrow may be an adaptation to the ephemeral, fire-mediated habitat that this species prefers. In recent times, high vagility also appears to have offset inbreeding and loss of genetic diversity in highly fragmented habitat.     

Differences in sperm migration through cervical mucus in vitro relates to sperm colonization of the oviduct and fertilizing ability in goats

Sperm migration in estrous cervical mucus can be used to measure the ability of spermatozoa to migrate through the genital tract. The relationship of this test with the sperm colonization of the isthmus, and its impact on fertility has not been evaluated in goats. Our objectives were to determine the differences among spermatozoa of different bucks in their ability to penetrate homologous cervical mucus in vitro and to determine the relationship between sperm displacement through cervical mucus and the ability of spermatozoa to colonize the oviduct and penetrate IVM oocytes, in vivo. Sperm migration in cervical mucus was assessed in flat capillary tubes with a phase contrast microscope. In the first experiment, fresh semen was used to establish differences between males in the ability of their spermatozoa to migrate in cervical mucus. In the second experiment, goats in estrus were inseminated with fresh spermatozoa from males with significant differences in mucus migration ability, and sperm numbers were evaluated at the UTJ. In the third experiment, the fertilization efficiency of IVM oocytes transferred to the oviduct of estrous females inseminated with semen from the same males as earlier, was used to assess the relationship between the mucus migration test and the in vivo fertilization performance of their spermatozoa. Spermatozoa from different males varies significantly in sperm migration efficiency in cervical mucus (15.5a +/- 1.2; 14.9a +/- 1.4; 17.5ab +/- 1.2; 17.0ab +/- 1.5; 19.7b +/- 1.2; 20.1b +/- 1.4 mm; media +/- S.E.M. for males A-F, respectively, P < 0.05). Spermatozoa from males with different mucus migration efficiency values produced different sperm populations at the oviduct reservoir of inseminated females (1,233 +/- 92.3 versus 28.8 +/- 17.0 spermatozoa of males with high and low relative migration efficiency, respectively, P < 0.02). Spermatozoa from males with different mucus migration efficiency values have different fertilization rates of IVM oocytes transferred to oviduct (47/96 (49.0%) versus 25/91 (27.5%) for males with high and low relative migration efficiency, respectively, P < 0.05). Cumulative results suggest that sperm migration in cervical mucus is related to the ability of spermatozoa to colonize the oviduct and to fertilize matured oocytes in vivo.     

Alkaline Phosphatase Content and the Effects of Prednisolone on Mammalian Cells in Culture

The alkaline phosphatase content of different tissue culture cell lines has been shown to vary from no detectable activity to high enzyme concentration. Within the epithelial lines studied alkaline phosphatase is either constitutive or inducible. Two epithelial cell strains in which alkaline phosphatase was "absent" could be induced to develop significant amounts of the enzyme when grown in the presence of Δ¹-hydrocortisone. Phosphate did not repress enzyme induction by prednisolone. Under conditions of deadaptation the induced enzyme was diluted by cell multiplication. The mouse fibroblastic L line and several human fibroblastic lines did not contain alkaline phosphatase when grown under the conditions described nor could they be induced to produce the enzyme when cultivated in medium with prednisolone. Δ¹-Hydrocortisone has other characteristic effects on established mammalian cell cultures which vary among cell lines. Human epithelial lines show reduction in cell multiplication with increase in mitotic index. The cytoplasm is increased and cell volume is nearly doubled. Mouse fibroblasts show a similar reduction in cell multiplication with a decrease in mitotic index. There is no increase in cell cytoplasm. Human fibroblast strains show no inhibition of multiplication or alteration in total cell protein when grown in medium containing prednisolone. Antisera prepared against "negative" prednisolone-inducible human cell lines and against a positive human line inhibited alkaline phosphatase activity to an equal degree.     

In vitro induction of suppressor cells by plasma of rats with Trypanosoma brucei rhodesiense infection

Mitogenic responses of B and T lymphocytes from spleens of rats infected with Trypanosoma brucei rhodesiense were suppressed. Plasma from infected rats suppressed the mitogenic responses of B and T lymphocytes from spleens of normal uninfected rats. Removal of immune complexes from plasma of infected rats significantly reduced the suppressive effect of the plasma on splenic lymphocytes of normal uninfected rats. Normal thymus cells treated with plasma from infected rats and added to cultures of normal spleen lymphocytes inhibited the mitogenic responses of B and T lymphocytes. We suggest that the interaction of immune complexes and Fc or C3b receptors of T lymphocytes resulted in the in vitro induction or activation of T suppressor lymphocytes.     

Mass Spectrometric Studies of the Effect of pH on the Accumulation of Intermediates in Denitrification by Paracoccus denitrificans

We have used a quadrupole mass spectrometer with a gas-permeable membrane inlet for continuous measurements of the production of N₂O and N₂ from nitrate or nitrite by cell suspensions of Paracoccus denitrificans. The use of nitrate and nitrite labeled with ¹⁵N was shown to simplify the interpretation of the results when these gases were measured. This approach was used to study the effect of pH on the production of denitrification intermediates from nitrate and nitrite under anoxic conditions. The kinetic patterns observed were quite different at acidic and alkaline pH values. At pH 5.5, first nitrate was converted to nitrite, then nitrite was converted to N₂O, and finally N₂O was converted to N₂. At pH 8.5, nitrate was converted directly to N₂, and the intermediates accumulated to only low steady-state concentrations. The sequential usage of nitrate, nitrite, and nitrous oxide observed at pH 5.5 was simulated by using a kinetic model of a branched electron transport chain in which alternative terminal reductases compete for a common reductant.     

Axonal guidance in the chick visual system: posterior tectal membranes induce collapse of growth cones from the temporal retina

Membranes from posterior and anterior thirds of the chick optic tectum were added to explants from nasal and temporal retina. Posterior membranes, and to a lesser extent anterior membranes, cause temporal growth cones to collapse and their axonal processes to retract. Neither tectal source has an effect on nasal growth cones. We interpret these results to mean that there is a tectal activity, stronger in the posterior than the anterior region of the tectum, which helps guide growth cones during the development of the retinotectal map. We believe that in vivo this activity helps to steer temporal growth cones away from the posterior tectum. Nasal growth cones, which must map to the posterior tectum, are resistant to it. In vitro, when posterior membranes contact temporal growth cones over their surface, filopodia and lamellipodia withdraw rapidly. This leads to loss of contact between the growth cone and the substrate, followed by collapse.