Oxytocin- and vasopressin-induced growth of human small-cell lung cancer is mediated by the mitogen-activated protein kinase pathway.

Malignant growth of small-cell lung carcinoma is promoted by various neuroendocrine autocrine/paracrine loops. Therefore, to interfere with this mitogenic process, it is crucial to elucidate the mechanisms involved. It is known that the oxytocin (OT) and vasopressin (VP) genes, normally transcriptionally restricted in their expression, are activated in small-cell lung cancer (SCLC), concomitantly with expression of their receptors (OTR, V1aR, V1bR/V3R and V2R). The aim of the present study was to characterize, in concentrations close to physiological and pharmacological conditions, intracellular signalling events triggered by OT and VP binding to their specific receptors in SCLC cells and to identify factors mediating OT- and VP-induced mitogenic effects on SCLC. Known agonists for OTR ([Thr4,Gly7]OT) and V1aR (F180), in addition to OT and VP, were able to elicit increases in cytosolic Ca2+ levels and this effect could be blocked using an OTR antagonist (OVTA) or a V1aR antagonist (SR49059) respectively. There was no activation of the cAMP pathway detected after VP, dDAVP (a V2R agonist), or OT treatment. Stimulation of SCLC cells with OT and VP led to an increase of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, maximal at 5 min, and the subsequent phosphorylation of its downstream target p90 ribosomal S6 kinase (p90RSK). Pre-incubation with OVTA and SR49059, and with inhibitors of phospholipase C (PLC), protein kinase C (PKC), mitogen-activated protein kinase/ERK kinase (MEK) 1/2 and a Ca2+ chelator significantly reduced OT- and VP-induced ERK1/2 phosphorylations. OVTA, SR49059 as well as MEK1/2 and PKC inhibitors also downregulated OT- and VP-induced p90RSK phosphorylation. In [3H]thymidine-uptake experiments, we subsequently observed that PLC, Ca2+, PKC and ERK1/2 are absolutely required for the OT- and VP-stimulated SCLC cellular growth process. In conclusion, the results presented here indicate that OT- and VP-induced mitogenic effects on SCLC are respectively mediated by OTR and V1aR signalling and that this mitogenic signalling passes through the phosphorylation of ERK1/2 and p90RSK in a PLC-, Ca2+-, PKC- and MEK1/2-dependent pathway.

Key peptide processing enzymes are expressed by breast cancer cells.

The expression of the three key peptide processing enzyme families, represented by CPE, PAM, and PC1/3 plus PC2, were examined in MCF-7 and ZR-75-1 breast cancer cell lines. Both of these cell lines express vasopressin receptors as well as the vasopressin gene, but the processing of vasopressin gene-related proteins appears to be limited. Products of the expected size for, CPE, PAM and PC1/PC3 could be amplified by reverse transcription-polymerase chain reaction (RT-PCR) from both cell lines. Cloning and sequencing of these RT-PCR products revealed that each enzyme mRNA had a structure identical to that published for the human form of the respective enzyme. Western analysis provided evidence that mRNAs for these enzymes are translated into proteins. Alternatively, PC2 mRNA was identified to be present in MCF-7 cells both by RT-PCR and Western blot analysis, but could not be demonstrated for ZR-75-1 cells. Our findings suggest that the key processing enzymes needed to generate active vasopressin and other neuropeptide growth factors are present in breast cancer cells.

The internalization and endosomal trafficking of the EGF receptor in response to EGF is delayed in the waved-2 mouse liver.

The EGF receptor in waved-2 mice contains a point mutation that renders it kinase-deficient. We investigated how the waved-2 mutation affects the internalization and endosomal trafficking of the receptor in vivo in response to EGF. When the waved-2 mice were injected with EGF, there was approximately 50% less tyrosine phosphorylation detected in whole-liver homogenate compared to wild-type background mice. Although EGF increased the EGF receptor levels in the early and late liver endosomal fractions of waved-2 mice, its trafficking was delayed compared to wild-type mice. Ubiquitination of the EGF receptor may affect its endosomal sorting. We found that a similar amount of EGF receptor was immunoprecipitated from the endosomal fractions of EGF-treated waved-2 and wild-type with anti-ubiquitin antibody. These results demonstrate that the waved-2 EGF receptor can become ubiquitinated and can be trafficked to the late endosomes, although it appears that its kinase deficiency delays this process.

Thyrotropin regulates the levels of free ubiquitin and ubiquitin-protein conjugates in the male mouse thyroid.

The conjugation of ubiquitin to proteins can be a signal for their degradation, but can also be involved in regulatory processes not directly involved in protein degradation. Because thyrotropin (TSH) is the major physiological regulator of the thyroid, we have investigated whether changes in the circulating level of TSH influence the level of immunoreactive ubiquitin in the thyroid, as assessed by dot-blot assays. Putting male Balb/c mice on a low iodine diet with methimazole (MMI) in their drinking water for 14 days raised the level of ubiquitin by 425 % (p < 0.001) per microgram nonthyroglobulin protein (the mean thyroglobulin level dropped by 35% (p < 0.05)). Western blots similarly indicated that immunoreactivity migrating in the region of monoubiquitin, ubiquitin oligomers, and ubiquitinated thyroid proteins increased on the low iodine/MMI diet. Injecting 1 microg triiodothyronine (T3) 6 hours prior to sacrifice appeared to reduce the ubiquitin levels by 31% when compared with mice only on the low iodine/MMI (p < 0.07), but injection of T3 had no effect on ubiquitin levels in mice on the control diet. Injecting male ICR mice with a large dose of TSH (200 mU) increased immunoreactive ubiquitin levels by 50% (p < 0.05) 2 hours later. After a second dose of TSH was injected 12 hours later, the level of immunoreactive thyroglobulin fell by 17% (p < 0.05). With further 12 hourly injections, thyroglobulin levels then began to reaccumulate, and they had returned to the level of saline-injected controls after 50 hours (five TSH injections), while ubiquitin levels fell, but remained significantly elevated above the saline-injected controls (36%, p < 0.01). When ICR mice were given perchlorate in their drinking water to block the iodide pump, thus preserving thyroidal responsiveness to repeated TSH injections, the responses to the initial two TSH injections were similar to mice who received ordinary tap water. However, with further TSH injections, the reaccumulation of thyroglobulin did not occur: at 50 hours, thyroglobulin levels remained suppressed by 28% (p < 0.05), and ubiquitin levels actually rose slightly, and were significantly higher (57%, p < 0.01) than the saline-injected controls. These in vivo responses of free ubiquitin, oligoubiquitin, and ubiquitinated protein to changes in the level of circulating TSH suggest that ubiquitin-mediated mechanisms are involved in some of the thyroid's metabolic responses.

The cytosolic 47-kilodalton protein that binds to the 3' untranslated region of epidermal growth factor transcripts responds to orchiectomy in a tissue-specific fashion.

The submaxillary gland (SMG) of the male mouse is known to contain more epidermal growth factor (EGF) messenger RNA (mRNA) than that of the female. In contrast, the SMG of female mice contains more of a cytosolic 47-kDa RNA binding protein that binds to a unique 23-b sequence at the end of the 3' untranslated region (3' UTR) of EGF, which includes the canonical polyadenylation signal in an AU-rich region containing a potential mRNA destabilizing sequence. Testosterone treatment can change the activity of this 47-kDa protein within 1 day, which precedes the change produced in the level and in the polyadenylation of EGF mRNA in female mice. In the kidney, however, neither the 47-kDa protein nor the polyadenylation pattern changed after testosterone. We now report that 2 weeks following orchiectomy of Balb/c mice, mature EGF peptide immunoreactivity levels fall by 96% (P < 0.0002), and mRNA levels fall by 76% (P < 0.0001). Orchiectomy also enhances the RNA binding activities of the 47-kDa protein in SMG cytosol but not in the kidney: UV cross-linking to 3' UTR RNA is increased by 67% (P < 0.01) and shifting of the gel mobility of the RNA is increased by 47% (P < 0.05). The polyadenylation pattern of EGF transcripts also changes in SMG but not in kidney cytosol. After orchiectomy, the population of EGF transcripts in the SMG with short, heterogenous poly-A tails (<50 A's) decreases by 31% (P < 0.03), whereas transcripts with long poly-A tails of approximately 50, 70, 100, and 200 A's all increase and become more distinct, resembling the pattern found in the normal female. Thus, 2 weeks after orchiectomy, the responses in EGF expression, EGF mRNA polyadenylation, and the activities of the 47-kDa 3' UTR binding protein are the reciprocal of the responses produced by injecting female mice with testosterone for 5 days, providing further evidence that circulating androgens regulate EGF expression posttranscriptionally in a sexually dimorphic, tissue-specific fashion.

Increased epidermal growth factor expression produced by testosterone in the submaxillary gland of female mice is accompanied by changes in poly-A tail length and periodicity.

The level of mature EGF messenger RNA (mRNA) in the female submaxillary salivary gland (SMG) begins to rise after 3 days' treatment with testosterone (200 micrograms sc qod), and by 5 days it reaches a plateau of approximately 5 times baseline. Testosterone can increase the transcription of other genes in the SMG rapidly, so the lag in the EGF is not due to a slow androgen receptor response, and EGF mRNA can respond rapidly to other mediators, so the lag is not an innate characteristic of EGF transcription. Immunoreactive EGF levels reach a steady-state several times greater than the plateau reached by EGF mRNA, suggesting that testosterone also enhances the efficiency of EGF mRNA translation. Because testosterone has been reported to alter poly-A polymerase activities and because the translation and stability of some mRNAs is affected by changes in their polyadenylation, we used 3' rapid amplification of complementary DNA ends (3' RACE) to determine whether testosterone affected this aspect of EGF RNA metabolism. We found that EGF transcripts in untreated female SMG occur indistinct size classes, with poly-A tails of approximately 20, 50, 70, 100, and 200 A's attached after the terminal polyadenylation signal. In contrast, EGF transcripts in male SMG have poly-A tails of less clearly defined lengths, being more heterogeneous, ranging from approximately 20-100 A's. Treating female mice with testosterone causes the poly-A pattern in the SMG to change to a more heterogeneous population ranging from approximately 20-100 A's, similar to the male pattern. This change in EGF transcript polyadenylation occurs concurrently with the changes observed in the levels of EGF mRNA. EGF transcripts from male or female kidney contain distinct poly-A tails of approximately 20, 50, 70, 100, and 200 A's: neither EGF mRNA levels nor polyadenylation was altered by testosterone. The tissue-specific increase in EGF mRNA levels and in translational efficiency produced by testosterone in the female mouse SMG could involve this posttranscriptional alteration in transcript polyadenylation.