Cancer cells have broken circadian clocks when compared to their normal tissue counterparts. Moreover, it has been shown in breast cancer that disruption of common circadian oscillations is associated with a more negative prognosis. Numerous studies, focused on canonical circadian genes in breast cancer cell lines, have suggested that there are no mRNA circadian-like oscillations. Nevertheless, cancer cell lines have not been extensively characterized and it is unknown to what extent the circadian oscillations are disrupted. We have chosen representative non-cancerous and cancerous breast cell lines (MCF-10A, MCF-7, ZR-75-30, MDA-MB-231 and HCC-1954) in order to determine the degree to which the circadian clock is damaged. We used serum shock to synchronize the circadian clocks in culture. Our aim was to initially observe the time course of gene expression using cDNA microarrays in the non-cancerous MCF-10A and the cancerous MCF-7 cells for screening and then to characterize specific genes in other cell lines. We used a cosine function to select highly correlated profiles. Some of the identified genes were validated by quantitative polymerase chain reaction (qPCR) and further evaluated in the other breast cancer cell lines. Interestingly, we observed that breast cancer and non-cancerous cultured cells are able to generate specific circadian expression profiles in response to the serum shock. The rhythmic genes, suggested via microarray and measured in each particular subtype, suggest that each breast cancer cell type responds differently to the circadian synchronization. Future results could identify circadian-like genes that are altered in breast cancer and non-cancerous cells, which can be used to propose novel treatments. Breast cell lines are potential models for in vitro studies of circadian clocks and clock-controlled pathways.

Cyclic nucleotide-gated channels (CNGC) open in response to the binding of 3'5'-cyclic nucleotides. Members of the CNGC family vary as much as 100-fold in their ability to respond to cAMP and cGMP. Molecular models of the nucleotide binding domains of the bovine retina and catfish and rat olfactory CNGCs were built from the crystal structure of cAMP bound to catabolite gene activator protein (CAP) with AMMP, a program for molecular mechanics and dynamics. The nucleotide conformation can be predicted from the number of strong and weak interactions between the purine ring and the binding site. The amino acids predicted to be important for determining the nucleotide affinity and specificity are residues 61, 83 (mediated through a water molecule), 119 and 127 (CAP sequence numbers) which interact with the purine ring. These residues also dictate the conformation of the ligand in the binding pocket. cGMP is preferentially bound in the syn conformation in bovine retina, bovine olfactory and rat olfactory CNGCs due to Thr83, while either conformation can bind in catfish olfactory CNGC. cAMP is predicted to bind either in syn or anti conformation, depending on the interaction with residue 119: the anti conformation is preferentially bound in olfactory CNGCs.

Loss of receptor hair cells in the cochlea accounts for a significant proportion of hearing impairment in the population. Hair cells can be lost as a consequence of viral or bacterial insult, aging, and damage from intense sound or aminoglycoside antibiotics. The generation of replacement hair cells following damage by sound or drugs has been clearly demonstrated in birds; the chick is the best-studied model for auditory hair cell regeneration. New hair cells arise as progeny from an otherwise nondividing supporting cell population induced to proliferate by the damage. Functional recovery of hearing accompanies this cellular recovery process. The signals and pathways responsible for regenerative proliferation are unknown. Here we show that proliferation is induced in the undamaged receptor epithelium by agents that increase cyclic AMP levels, and that following this stimulation hair cells become labeled with proliferation markers. This remarkable proliferative response is blocked by inhibitors of the cAMP-regulated protein kinase A (PKA). In addition we show that the proliferative response induced by in vitro gentamicin damage is also significantly blocked by PKA inhibitors. These observations are the first to identify a signaling pathway that plays a role in regenerative proliferation in the auditory receptor epithelium.

The Escherichia coli cAMP receptor protein (CRP) displays biphasic characteristics in protease and beta-galactosidase induction assays at increasing cAMP concentrations in response to ligand binding at the secondary binding site located between the primary binding site and the DNA binding domain. Two mutants were created to determine the mechanistic reason for the CRP biphasic response by inhibiting binding of cAMP to the secondary site via interference with the Arg 181 interaction with the ligand's phosphate. The S179A/R180D/E181H mutant binds two cAMP molecules per dimer, does not exhibit a biphasic response, lacks selective DNA binding, and has inhibited nonselective DNA binding. The R180K mutant binds four cAMP molecules per dimer, exhibits a biphasic response, nonselective DNA binding similar to CRP, but has inhibited selective DNA binding characteristics. The results are consistent with a 2 x 2-binding site scheme were both primary binding sites must be occupied before the secondary binding sites are occupied. A structural mechanism suggesting the secondary sites are formed by binding of cAMP to the primary sites is proposed. AMMP-generated molecular models suggest that R180 orients E181 to produce selective DNA binding, Arg 169 interactions are necessary for nonselective DNA binding, and the position of Leu 57 inhibits chymotrypsin cleavage of Phe 136. DNA binding results suggest that CRP may be the unknown transcription factor which binds to the temperature sensitive dsrA promoter.

Photoreceptor outer segments transduce information about incoming light levels through a class of ion channels that respond directly to changes in cytosolic 3',5'-cyclic guanosine monophosphate levels. A series of 3',5'-cyclic purine analogues with alterations at N1, C2, C6, or C8 positions was used to examine molecular interactions between the nucleotide and the channel. The maximal current activated by C2-altered analogues in excised membrane patches was less than the current activated by cGMP, and the K0.5, the concentration which activates 50% of the current in a patch, was increased. Nonpolar C8-substituted cAMP analogues activated more current than the parent cAMP with lower K0.5 values. This was in contrast to 8-amino-cAMP, which exhibited greatly reduced activity. The rank order of activity, based on K0.5 values, for C8-cAMP substituents was as follows: 8-azido- > 8-methylamino- > 8-benzylamino- > cAMP > 8-bromo- > 8-hydroxy- >> 8-amino-cAMP. 1,N6-Etheno-cAMP and N6-monobutyryl-cAMP activated a small fraction of the total possible current with high K0.5 values. Other analogues with alterations at N1 or C6 positions including N1-oxide-cAMP, 2-aminopurine riboside 3',5'-monophosphate, and N6-monosuccinyl-cAMP do not bind to the channel, suggesting that interactions with the channel in this region are essential for binding. In order to help interpret the changes in maximal current and K0.5 values compared to cGMP, molecular models of the active analogues were constructed and then docked into a molecular model of the cyclic nucleotide binding site of the retinal channel. This model, proposed by Kumar and Weber [(1992) Biochemistry 31, 4643-4649], was based on the crystal structure of cAMP bound to catabolite activator protein. Our modeling showed that the analogues were sterically accommodated within the binding site. No hydrogen bonds were predicted between the purine rings of cAMP and the pocket; however, Phe 533 on the beta 5 strand was predicted to form weak electrostatic interactions with C6 substituents on both cAMP and cGMP. The importance of contacts in this region of the binding pocket is further emphasized by the inactive analogues, all of which are altered at N1 or C6.