Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells.

Cellular growth and differentiation are controlled by multiple extracellular signals, many of which activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases. Components of the MAP kinase pathways also cause oncogenic transformation in their constitutively active forms. Moreover, expression of activated ras can confer metastatic potential upon some cells. Activation of MAP kinases requires phosphorylation of both Thr and Tyr in the catalytic domain by a family of dual-specificity kinases, called MEKs (MAP kinase/ERK kinase). MEK1 is activated by phosphorylation at Ser218 and Ser222 by Raf. Mutation of these two sites to acidic residues, specifically [Asp218], [Asp218, Asp222], and [Glu218, Glu222], results in constitutively active MEK1. Using these mutant variants of MEK1, we showed previously that transfection of NIH/3T3 or Swiss 3T3 cells causes morphological transformation and increases growth on soft agar, independent of ERK activity. The transformed cell lines show increased expression of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells transfected with the [Asp218] or [Asp218, Asp222] for metastatic potential after i.v. injection into athymic mice. Parental 3T3 cells formed no tumors grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metastases. Thus, like activated Ras, MEK1 can confer both tumorigenic and metastatic potential upon NIH3T3 cells. These results refine the mechanism through which ras could confer tumorigenic and metastatic potential (ie., the critical determinants of tumorigenic and metastatic potential are downstream of MEK1).

Analysis of mechanisms underlying BRMS1 suppression of metastasis.

Introduction of normal, neomycin-tagged human chromosome 11 (neo11) reduces the metastatic capacity of MDA-MB-435 human breast carcinoma cells by 70-90% without affecting tumorigenicity. Differential display comparing MDA-MB-435 and neo11/435 led to the discovery of a human breast carcinoma metastasis suppressor gene, BRMS1, which maps to chromosome 11q13.1-q13.2. Stable transfectants of MDA-MB-435 and MDA-MB-231 breast carcinoma cells with BRMS1 cDNA still form progressively growing, locally invasive tumors when injected in mammary fat pads of athymic mice but exhibit significantly lower metastatic potential (50-90% inhibition) to lungs and regional lymph nodes. To begin elucidating the mechanism(s) of action, we measured the ability of BRMS1 to perturb individual steps of the metastatic cascade modeled in vitro. Consistent differences were not observed for adhesion to extracellular matrix components (laminin, fibronectin, type IV collagen, type I collagen, Matrigel); growth rates in vitro or in vivo; expression of matrix metalloproteinases, heparanase, or invasion. Likewise. BRMS1 expression did not up regulate expression of other metastasis suppressors, such as NM23, Kai1, KiSS1 or E-cadherin. Motility of BRMS1 transfectants was modestly inhibited (30-60%) compared to parental and vector-only transfectants. Ability to grow in soft agar was also decreased in MDA-MB-435 cells by 80-89%, but the decrease for MDA-MB-231 was less (13-15% reduction). Also, transfection and re-expression of BRMS1 restored the ability of human breast carcinoma cells to form functional homotypic gap junctions. Collectively, these data suggest that BRMS1 suppresses metastasis of human breast carcinoma by complex, atypical mechanisms.

Pontine nitric oxide modulates acetylcholine release, rapid eye movement sleep generation, and respiratory rate.

Pontine cholinergic neurotransmission is known to play a key role in the regulation of rapid eye movement (REM) sleep and to contribute to state-dependent respiratory depression. Nitric oxide (NO) has been shown to alter the release of acetylcholine (ACh) in a number of brain regions, and previous studies indicate that NO may participate in the modulation of sleep/wake states. The present investigation tested the hypothesis that inhibition of NO synthase (NOS) within the medial pontine reticular formation (mPRF) of the unanesthetized cat would decrease ACh release, inhibit REM sleep, and prevent cholinergically mediated respiratory depression. Local NOS inhibition by microdialysis delivery of N(G)-nitro-L-arginine (NLA) significantly reduced ACh release in the cholinergic cell body region of the pedunculopontine tegmental nucleus and in the cholinoceptive mPRF. A second series of experiments demonstrated that mPRF microinjection of NLA significantly reduced the amount of REM sleep and the REM sleep-like state caused by mPRF injection of the acetylcholinesterase inhibitor neostigmine. Duration but not frequency of REM sleep epochs was significantly decreased by mPRF NLA administration. Injection of NLA into the mPRF before neostigmine injection also blocked the ability of neostigmine to decrease respiratory rate during the REM sleep-like state. Taken together, these findings suggest that mPRF NO contributes to the modulation of ACh release, REM sleep, and breathing.

Nitric oxide synthase inhibition decreases pontine acetylcholine release.

This study tested the hypothesis that inhibition of nitric oxide synthase (NOS) in the medial pontine reticular formation (mPRF) would cause decreased acetylcholine (ACh) release. Microdialysis of cat mPRF permitted measurement of ACh during states of wakefulness, non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep. ACh release during microdialysis with Ringers (control) was compared to ACh release during microdialysis with 10 mM NG-nitro-L-arginine (NLA). The NOS inhibitor NLA caused a significant reduction in ACh released from the mPRF during wakefulness, NREM sleep, and REM sleep. This reduction in mPRF ACh release elicited by NLA suggests that nitric oxide (NO) contributes to cholinergic neurotransmission in the pontine reticular formation.