MDR1 causes resistance to the antitumour drug miltefosine

Miltefosine (hexadecylphosphocholine) is used for topical treatment of breast cancers. It has been shown previously that a high percentage of breast carcinomas express MDR1 or MRP. We investigated the sensitivity of MDR1 -expressing cells to treatment with miltefosine. We show that cells overexpressing MDR1 (NCI/ADR-RES, KB-8-5, KB-C1, CCRF/VCR1000, CCRF/ADR5000) were less sensitive to miltefosine treatment when compared to the sensitive parental cell lines. HeLa cells transfected with MDR1 exhibited resistance to the compound, indicating that expression of this gene is sufficient to reduce the sensitivity to miltefosine. The resistance of MDR1 -expressing cells to miltefosine was less pronounced than that to adriamycin or vinblastine. Expression of MDR2 did not correlate with the resistance to miltefosine. As shown by a fluorescence quenching assay using MIANS-labelled P-glycoprotein (PGP), miltefosine bound to PGP with a K d of approximately 7 μM and inhibited PGP-ATPase activity with an IC 50 of approximately 35 μM. Verapamil was not able to reverse the resistance to miltefosine. Concentrations of miltefosine up to approximately 60 μM stimulated, whereas higher concentrations inhibited the transport of [3H]-colchicine with an IC 50 of approximately 297 μM. Binding studies indicated that miltefosine seems to interact with the transmembrane domain and not the cytosolic nucleotide-binding domain of PGP. These data indicate that expression of MDR1 may reduce the response to miltefosine in patients and that this compound interacts with PGP in a manner different from a number of other substrates. © 2001 Cancer Research Campaign www.bjcancer.co

Rap1, a small GTP-binding protein is upregulated during arrest of proliferation in human keratinocytes

Rap1 is a small GTP-binding protein (SMG) that exists in two 95% homologous isoforms, rap1A and rap1B. The functions of the rap1 proteins are not well understood. In this report we examined expression and function of rap1 in primary (HOKs) and immortalized (IHOKs) human oral keratinocytes under different growth conditions. In HOKs, rap1 increased with passage number, suggesting a role in differentiation and arrest of proliferation. Similarly, when inhibition of proliferation and differentiation were induced in HOKs by 1.2 mM CaCl 2 , both rap1 and involucrin increased with increasing concentrations of CaCl 2 . However, when similar experiments were done with IHOKs, which continue to proliferate in the presence of 1.2 mM CaCl 2 , the increase in involucrin expression was similar to HOKs but there was no substantial increase in rap1, suggesting that increased expression of rap1 is linked to inhibition of proliferation rather than differentiation of keratinocytes. Upon transfection of immortalized keratinocytes with rapGAP, which inactivates both isoforms of endogenous active rap1, enhanced proliferation was observed. Thus, we conclude that rap1 inhibits proliferation in keratinocytes. J. Cell. Physiol. 196: 532–540, 2003. © 2003 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34444/1/10331_ftp.pd

Captive breeding of European freshwater mussels as aconservation tool: A review

1. Freshwater mussels are declining throughout their range. Their importantecological functions along with insufficient levels of natural recruitment haveprompted captive breeding for population augmentation and questions about the usefulness and applicability of such measures. 2. This article reviews the current state of captive breeding and rearing programmes for freshwater mussels in Europe. It considers the various species, strategies, andtechniques of propagation, as well as the different levels of effort requiredaccording to rearing method, highlighting the key factors of success. 3. Within the last 30 years, 46 breeding activities in 16 European countries have been reported, mainly of Margaritifera margaritifera and Unio crassus. Some facilities propagate species that are in a very critical situation, such as Pseudunio auricularius, Unio mancus, and Unio ravoisieri, or multiple species concurrently. Insome streams, the number of released captive-bred mussels already exceeds the size of the remaining natural population. 4. Rearing efforts range from highly intensive laboratory incubation to lowerintensity methods using in-river mussel cages or silos. Most breeding efforts are funded by national and EU LIFE(+) grants, are well documented, and consider the genetic integrity of the propagated mussels. Limited long-term funding perspectives, the availability of experienced staff, water quality, and feeding/survival during early life stages are seen as the most important challenges. 5. Successful captive breeding programmes need to be combined with restoration ofthe habitats into which the mussels are released. This work will benefit from anevidence-based approach, knowledge exchange among facilities, and an overall breeding strategy comprising multiple countries and conservation units. aquaculture, captive breeding, conservation translocation, freshwater mussel culturing, Margaritifera margaritifera, propagation, reintroduction, Unio crassusCaptive breeding of European freshwater mussels as aconservation tool: A reviewpublishedVersio