Microtubules are linear polymers of ␣-and ␤-tubulin heterodimers and are the major constituents of mitotic spindles, which are essential for the separation of chromosomes during mitosis. Here we describe a synthetic compound, 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (T138067), which covalently and selectively modifies the ␤ 1 , ␤ 2 , and ␤ 4 isotypes of ␤-tubulin at a conserved cysteine residue, thereby disrupting microtubule polymerization. Cells exposed to T138067 become altered in shape, indicating a collapse of the cytoskeleton, and show an increase in chromosomal ploidy. Subsequently, these cells undergo apoptosis. Furthermore, T138067 exhibits cytotoxicity against tumor cell lines that exhibit substantial resistance to vinblastine, paclitaxel, doxorubicin, and actinomycin D. T138067 is also equally efficacious in inhibiting the growth of sensitive and multidrug-resistant human tumor xenografts in athymic nude mice. These observations suggest that T138067 may be clinically useful for the treatment of multidrug-resistant tumors.

A number of studies in recent years have linked polymorphisms within the serotonin transporter (5HTT) gene to affective disorders and anxiety traits. The human 5HTT mRNA is alternatively spliced, and the splice variants are equally expressed in the human placental cell line and dorsal raphe. In this study, using 5¢ rapid amplification of cDNA ends, we show that the rat 5HTT mRNA is alternatively spliced, leading to three distinct mRNAs differing in the 5¢ untranslated region. To determine whether the three alternatively spliced mRNA species that contain one of the following untranslated regions (i) exon 1A, 63 bp (ii) exon 1A + 1B, 125 bp or (iii) exon 1C, 101 bp, were expressed in a tissue-specific manner, we used RT-PCR and exon-specific oligonucleotide hybridization. Our results suggest two of the variants (1A + 1B and 1A) may utilize the same promoter; however, they are not equally expressed. While in the adult CNS and adrenal medulla, the shorter mRNA consisting of exon 1A was considerably more abundant, in the stomach and heart, the two variants were equally expressed. The third splice variant exon 1C is only expressed in the gut and to a lesser extent in the heart. The data from this study suggest the splice variant consisting of exon 1C may utilize a distinct promoter compared to the other two. Keywords: alternative splicing, gut, promoter, serotonin transporter, tissue-specific. Serotonin (5HT) has been proposed to play a significant role in a number of physiological mechanisms such as sleep, appetite, thermoregulation, pain perception, and proper functioning of the bowel. Following vesicular release, 5HT is actively cleared from extracellular spaces by the serotonin transporter (5HTT or SERT). 5HTT has been shown to play an important role in a number of psychiatric disorders and in irritable bowel syndrome. Effective treatment of these disorders involves blocking 5HTT. Some of the psychiatric disorders have been linked to a number of polymorphisms within the 5HTT gene and its promoter (Collier et al. 1996;Lesch et al. 1996;Sander et al. 1997;Furlong et al. 1998;Greenberg et al. 2000;Twitchell et al. 2001). A recent study shows that the promoter of 5HTT may exhibit in vivo instability (Flattem and Blakely 2000).In the rodent and the human brain, 5HTT mRNA is predominantly localized to the raphe nuclei in the brain stem (for review see Hoffman et al. 1998). However, using RT-PCR, Lesch et al. (1993) and Ozsarac et al. (1998) detected low levels of the mRNA in the rat cortex. 5HTT uptake assays have indicated the presence of the transporter in primary astrocyte cultures, though clear evidence for its presence in vivo is lacking (Kimelberg and Katz 1985). Northern blot analysis of samples from the adult rat peripheral tissues showed that the 5HTT mRNA is present in the gut and the placenta, while absent in the heart and the liver (Chang et al. 1996;Hoffman et al. 1998).The serotonin transporter has been proposed to play a role in serotonergic neurotransmission and in the initiation of peristaltic a...

The novel anticancer compound T138067 is an irreversible inhibitor of tubulin polymerization. Amides 3-6 were synthesized using standard methodologies and determined to be significantly less lipophilic than T138067 based on logP calculations. Tubulin polymerization and [(3)H]-T138067 competition assays revealed that these amides are pro-drugs for parent aniline 2. Amides 3-5 showed no detectable signs of crossing the blood brain barrier, while amide 6 was found in extremely small amounts (12 ng/g of brain tissue). Aniline 2, which was formed in vivo from these amides, was found in significantly smaller amounts (approximately 20 to >5000 times) in the brain than when 2 was administered directly. The in vivo efficacy of amide 6 approached that of T138067 and was better tolerated when administered to athymic nude mice bearing MX-1 human mammary tumor xenografts.

We have been studying the molecular mechanism of neuronal differentiation through which the multipotent precursor becomes limited to the final transmitter phenotype. Here we focused on the role of the 5' proximal regulatory cassette (-190; +53 bp) of the rat enkephalin (rENK) gene in the developmental regulation of the enkephalin phenotype. Several well characterized cis-elements, including AP2, CREB, NF1, and NFkB, reside on this region of the rENK gene. These motifs were sufficient to confer activity-dependent expression of the gene during neurodifferentiation when it was tested using transient transfection assays of primary developing spinal cord neurons treated with tetrodotoxin (TTX). This region was then used as a DNA probe in mobility shift assays, with nuclear proteins derived from phenotypically and ontogenetically distinct brain regions. Only a few low abundance protein-DNA complexes were detected and only with nuclear proteins derived from developing but not from adult brain. The spatiotemporal pattern of these complexes did not show correlation with enkephalin expression which was assessed by RT-PCR. We employed synthetic probes corresponding to consensus as well as ENK-specific sequences of the individual motifs to identify the nature of the observed bands. Although both consensus NF1 and enkCRE1(NF1) formed complexes with nuclear proteins derived from the striatum and cortex at various ages, the appearance of the bands was not correlated with ENK expression. Surprisingly, no complexes were detected if other ENK-specific motifs were used as probes. We also tested nuclear extracts derived from forskolin-induced and control C6 glioma cells, again using the whole proximal regulatory cassette as well as individual motifs. These experiments showed the formation of elaborate protein-DNA bands. There was no direct correlation between the appearance of bands and forskolin-induced ENK expression. Unexpectedly, all ENK-specific motifs formed specific and highly abundant protein-DNA complexes when nuclear extracts from the human tumor cell line (HeLa), which does not express ENK, were used. Based on these observations, we concluded that: 1. Interactions between the proximal regulatory cassette and additional probably far distant regions of the rENK gene and their binding proteins may be necessary to confer developmentally regulated, cell-specific expression of the ENK gene; and 2. Inducibility of the gene by common cis-elements can be governed by this region; however, the cell-specificity of the induction remains elusive.