Differential regulation of MAP kinase signalling by dual-specificity protein phosphatases.

The regulated dephosphorylation of mitogen-activated protein kinases (MAPKs) plays a key role in determining the magnitude and duration of kinase activation and hence the physiological outcome of signalling. In mammalian cells, an important component of this control is mediated by the differential expression and activities of a family of 10 dual-specificity (Thr/Tyr) MAPK phosphatases (MKPs). These enzymes share a common structure in which MAPK substrate recognition is determined by sequences within an amino-terminal non-catalytic domain whereas MAPK binding often leads to a conformational change within the C-terminal catalytic domain resulting in increased enzyme activity. MKPs can either recognize and inactivate a single class of MAP kinase, as in the specific inactivation of extracellular signal regulated kinase (ERK) by the cytoplasmic phosphatase DUSP6/MKP-3 or can regulate more than one MAPK pathway as illustrated by the ability of DUSP1/MKP-1 to dephosphorylate ERK, c-Jun amino-terminal kinase and p38 in the cell nucleus. These properties, coupled with transcriptional regulation of MKP expression in response to stimuli that activate MAPK signalling, suggest a complex negative regulatory network in which individual MAPK activities can be subject to negative feedback control, but also raise the possibility that signalling through multiple MAPK pathways may be integrated at the level of regulation by MKPs.

Influence of beta1 integrins on epidermal squamous cell carcinoma formation in a transgenic mouse model: alpha3beta1, but not alpha2beta1, suppresses malignant conversion.

Although aberrant integrin expression has been documented in many epithelial tumors, little is known about how integrins influence neoplastic progression. To examine this issue, transgenic mice in which the alpha2beta1 or alpha3beta1 integrin was expressed in the suprabasal epidermal layers via the involucrin promoter were subjected to skin carcinogenesis. Equal numbers of benign squamous papillomas were observed in transgenic and wild-type animals. However, the frequency of conversion of papillomas to malignant squamous cell carcinomas was much lower in alpha3beta1 transgenic than in alpha2beta1 transgenic and wild-type mice. No differences were observed in apoptosis or in the expression of endogenous integrins in transgenic and wild-type papillomas. However, alpha3beta1 transgenic papillomas displayed a diminished proliferative capacity and were more highly differentiated as judged by BrdUrd incorporation and keratin 10 expression, respectively, than alpha2beta1 transgenic and wild-type papillomas. Two proteins that associate with alpha3beta1 and not alpha2beta1 are extracellular matrix metalloproteinase inducer and CD81. Extracellular matrix metalloproteinase inducer expression correlated inversely with the degree of differentiation in normal epidermis and in transgenic and wild-type papillomas. Up-regulation of CD81 was observed in 100% of wild-type and 88% of alpha2beta1 transgenic papillomas but in only 25% of alpha3beta1 transgenic papillomas. CD81 was undetectable in untreated epidermis and strongly expressed in all transgenic and wild-type squamous cell carcinomas. Our results demonstrate that the alpha3beta1 integrin can suppress malignant conversion, and that the mechanism may involve CD81.

Expression of involucrin in normal, hyperproliferative and neoplastic mouse keratinocytes.

Involucrin is a protein precursor of the epidermal cornified envelope. Although expression of the human protein has been documented extensively, studies in the mouse have been hampered by a shortage of good antibodies. We describe the production of recombinant mouse involucrin and preparation of rabbit antisera to the protein that work well by immunohistochemistry and Western blotting. We confirm that in normal mouse epidermis the onset of involucrin expression is in the upper spinous layers and inner root sheath of the hair follicle. Involucrin was also detected in the differentiating epithelial cells of normal tongue, oesophagus and bladder. Involucrin was expressed in a subpopulation of mouse keratinocytes cultured in standard or low calcium medium and the proportion of involucrin-positive cells increased during suspension-induced terminal differentiation. Western blotting of keratinocytes from several inbred mouse strains revealed a remarkable heterogeneity in the electrophoretic mobility of involucrin, reflecting inter-strain variation in the number of tandem repeats in the protein. In the hyperproliferative epidermis of healing wounds involucrin was expressed in most of the suprabasal layers. In epidermal papillomas and carcinomas involucrin expression correlated well with degree of histological differentiation. The sites of expression of the mouse protein were thus the same as those previously reported for human involucrin. With the development of the new antibodies we anticipate that involucrin will become as widely used a marker of keratinocyte differentiation in the mouse as it is in the human.

A multihit, multistage model of chemical carcinogenesis.

Carcinogenesis involves the accumulation of genetic changes within a single cell. Tumor promotion functions in the initial clonal expansion of an initiated cell but is generally not considered to influence later stages. To investigate whether tumor promotion can influence later stages of carcinogenesis we developed a two-hit 7, 12-dimethylbenz[a]anthracene (D) protocol designed to enrich for keratinocytes that contain at least two D-induced genetic alterations. FVB/N mice were initiated with D and promoted with 12-O-tetradecanoylphorbol-13-acetate (T) or treated with acetone (A) vehicle for 6 weeks. At 7 weeks after the start of promotion, but before visible papilloma development, groups of mice were treated with a second dose of D or A and 1 week later T promotion was resumed. D/T/A/T mice developed 2.8 papillomas/mouse and D/A/D/T mice demonstrated an additive tumor response and developed 5.8 papillomas/mouse. Importantly, D/T/D/T mice developed 12.4 papillomas/mouse, thereby demonstrating a synergistic tumor response compared with D/A/D/T and D/T/A/T mice. D/T/D/T papillomas exhibited increases in suprabasal S phase cells and keratin 13 expression when compared with D/T/A/T papillomas. D/T/D/T mice developed squamous cell carcinomas (SCCs) 10 weeks earlier than D/T/A/T mice and demonstrated a 96% malignancy incidence and 1.71 SCC/mouse compared with D/T/A/T mice, which demonstrated a 28% malignancy incidence and 0.32 SCC/mouse. Greater than 90% of D/T/A/T and D/T/D/T papillomas and SCCs contained mutant Ha-ras, while a normal Ha-ras allele persisted in all cases, indicating that a gene other than the remaining normal allele of Ha-ras was a target gene for the second D hit. These data demonstrate that: (i) promotion between the first and second hits has a profound outcome on carcinogenesis, presumably by increasing the probability that a second hit will occur in a previously initiated cell; (ii) continued promotion after the second hit is required for full expression of malignancy; (iii) the classic initiation-promotion protocol can be extended to a multihit, multistage model.

Mice deficient in tumor necrosis factor-alpha are resistant to skin carcinogenesis.

Given the associations between chronic inflammation and epithelial cancer, we studied susceptibility to skin carcinogenesis in mice deficient for the pro-inflammatory cytokine TNF-alpha (refs. 5,6). TNF-alpha(-/-) mice were resistant to development of benign and malignant skin tumors, whether induced by initiation with DMBA and promotion with TPA or by repeated dosing with DMBA. TNF-alpha(-/-) mice developed 5-10% the number of tumors developed by wild-type mice during initiation/promotion and 25% of those in wild-type mice after repeated carcinogen treatment. TNF-alpha could influence tumor and stromal cells during tumor development. The early stages of TPA promotion are characterized by keratinocyte hyperproliferation and inflammation. These were diminished in TNF-alpha(-/-) mice. TNF-alpha was extensively induced in the epidermis, but not the dermis, in TPA-treated wild-type skin, indicating that dermal inflammation is controlled by keratinocyte TNF-alpha production. Deletion of a TNF-alpha inducible chemokine also conferred some resistance to skin tumor development. TNF-alpha has little influence on later stages of carcinogenesis, as tumors in wild-type and TNF-alpha(-/-) mice had similar rates of malignant progression. These data provide evidence that a pro-inflammatory cytokine is required for de novo carcinogenesis and that TNF-alpha is important to the early stages of tumor promotion. Strategies that neutralize TNF-alpha production may be useful in cancer treatment and prevention.

Localization and expression of cornifin-alpha/SPRR1 in mouse epidermis, anagen hair follicles, and skin neoplasms.

Recently, cornifin-alpha/SPPR1 has been identified as a putative precursor protein of the cornified cell envelope. In this study, the expression and localization of cornifin-alpha/SPPR1 was examined in untreated and tumor promoter-treated mouse skin, hair follicles, and skin neoplasms. Western analysis with antiserum (SQ37A) to a rabbit cornifin-alpha peptide or antiserum (SQ37C) to a human SPRR1 peptide demonstrated a 31-kDa immunoreactive protein in mouse epidermis and Northern analysis revealed the presence of a 1-kb mRNA. Immunohistochemical staining of mouse skin with SQ37A or SQ37C revealed intense and specific staining of the infundibulum, isthmus, and of Henle's layer of the inner root sheath of the lower anagen hair follicle and weak staining of the telogen follicle and the suprabasal layers of the epidermis. Treatment of mouse skin with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) produced a large increase in cornifin-alpha/SPRR1 protein and mRNA. Immunohistochemical localization of cornifin-alpha/SPRR1 in TPA-treated skin indicated that cornifin-alpha/SPRR1 was increased in the suprabasal epidermis but not in the follicle. sn-1,2,-didecanoylglycerol, a model lipid second messenger, produced an increase in cornifin-alpha/SPRR1 protein similar to that of TPA, while mirex, a non-phorbol ester-type promoter had no effect. Topical doses of retinoic acid did not repress TPA-induced cornifin-alpha/SPRR1 expression. Papillomas demonstrated a 10- and 100-fold increase in cornifin-alpha/SPRR1 protein and mRNA, and expression was restricted to suprabasal cells. Squamous cell carcinomas exhibited an intermediate level of cornifin-alpha protein, and expression was restricted to keratinized areas. These data indicate: i) cornifin-alpha/SPRR1 is expressed in mouse skin; ii) cornifin-alpha/SPRR1 is localized to specific areas of the anagen hair follicle with weak staining in the telogen follicle and epidermis; iii) epidermal cornifin-alpha/SPRR1 expression is induced by phorbol ester and sn-1,2-didecanoylglycerol but not mirex, and iv) papillomas and squamous cell carcinomas demonstrate a constitutive increase in cornifin-alpha/SPRR1 in differentiated areas of the neoplasms.

Characterization of olfactory deficits in the rat following administration of 2,6-dichlorobenzonitrile (dichlobenil), 3,3'-iminodipropionitrile, or methimazole.

The histopathology of the olfactory mucosal lesion associated with ip administration of 2,6-dichlorobenzonitrile (dichlobenil) and 3,3'-iminodipropionitrile (IDPN) has been well documented. Whether there is an olfactory deficit associated with the partial loss of the olfactory mucosa (localized around the dorsal medial meatus of the nasal cavity) has yet to be determined. Dichlobenil (100 mg/kg) or IDPN (200 mg/kg) was administered ip to adult male Long-Evans rats previously trained in an olfactory task to find a food pellet buried in approximately 7.5 cm of bedding in a 0.61 x 1.2 x 0.61-m Plexiglass chamber. As a positive control, another group received 300 mg/kg ip of 1-methyl-2-mercaptoimidazole (methimazole), a dosing regimen which destroys nearly all of the olfactory mucosa. All three compounds caused a transient increase in the mean latency to find the pellet, with the magnitude of the effect positively correlated with the extent of the olfactory lesion. In order to determine whether these deficits resulted from olfactory dysfunction or impaired cognitive function (a deficit previously attributed to IDPN exposure), another group of rats was dosed as above and tested in another spatial memory task, the Morris water maze (MWM), which is less dependent upon olfactory function. No performance deficit was detected in the MWM. These data suggest that the transient olfactory deficit in the dichlobenil-, IDPN-, and methimazole-treated rats is attributable to defective olfactory function.

Distribution of microsomal epoxide hydrolase and glutathione S-transferase in the rat olfactory mucosa: relevance to distribution of lesions caused by systemically-administered olfactory toxicants.

This study represents part an of ongoing effort to understand the mechanism underlying the distribution of the olfactory mucosal lesion resulting from the systemic administration of compounds such as 2,6-dichlorobenzonitrile (dichlobenil) and beta,beta'-iminodipropionitrile (IDPN). Immunohistochemistry was performed to localize the microsomal form of epoxide hydrolase (mEH) and glutathione S-transferase (GST) isozymes alpha, mu and pi in the rodent olfactory mucosa. GST-pi was found in abundance in the Bowman's glands of the mucosa lining the dorsal medial meatus (DMM) of the nasal cavity and in the nuclei of basal and sustentacular cells of the dorsal and lateral nasal cavity. Liver and olfactory mucosal levels of mEH are equivalent by Western blot analysis. mEH appeared to be localized in the apical cytoplasm of sustentacular cells in all regions of the olfactory mucosa except for the epithelium lining the DMM. These observations, coupled with the known profile of metabolites for dichlobenil, suggest that systemically-administered compounds causing site-specific lesions in the epithelium lining the DMM of the nasal cavity may do so by the in situ production of reactive epoxide metabolites which are then poorly capable of being detoxified. Thus, the distribution of metabolic enzymes, rather than the absolute level of an enzyme in a tissue, may dictate lesion distribution in the case of toxicants which are bioactivated in target tissues.

Genetic alterations cooperate with v-Ha-ras to accelerate multistage carcinogenesis in TG.AC transgenic mouse skin.

TG.AC transgenic mice harbor a v-Ha-ras transgene and retain two normal c-Ha-ras alleles and are susceptible to skin tumor formation by 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine whether normal c-Ha-ras antagonizes the oncogenic potential of the v-Ha-ras transgene and/or whether additional non-Ha-ras 7,12-dimethylbenz(a)anthracene (DMBA) initiation target genes exist in mouse skin, which could cooperate with v-Ha-ras to increase the frequency of initiation, rate of promotion, or risk of malignant conversion, we treated TG.AC mouse skin with a single subthreshold dose of DMBA. This was followed by limited TPA or diacylglycerol promotion to select for cells with additional genetic alterations over those cells containing the v-Ha-ras transgene only. DMBA-treated/TPA-promoted TG.AC mice demonstrated a 10-fold increase in the average number of papillomas per mouse, a greater incidence of papilloma bearing-mice, and an increased papilloma growth rate when compared to acetone-treated/TPA-promoted TG.AC mice. These profound changes in papilloma frequency and growth occurred in the absence of the characteristic DMBA-induced A182-->T mutation in c-Ha-ras and immunohistochemical nuclear staining for p53 protein. DMBA-treated/acetone-promoted TG.AC mice did not develop any tumors. Limited promotion with the model diacylglycerol, sn-1,2-didecanoylglycerol, similarly produced an average of 10-fold more papillomas in DMBA-treated mice than in acetone-treated/sn-1,2-didecanoylglycerol-promoted TG.AC mice. DMBA-treated/TPA-promoted TG.AC mice developed their first malignancy by 16 weeks, and by 30 weeks, 50% of the mice developed malignancies, whereas no malignancies were observed in acetone-treated/TPA-promoted TG.AC mice. These results indicate that there exist unidentified DMBA initiation target genes in TG.AC mouse skin that cooperate with mutant Ha-ras to increase papilloma frequency, growth, and malignant conversion, and that promoter treatment can influence malignant conversion by selecting for cells with multiple genetic alterations.

The irritable bowel syndrome: long-term prognosis and the physician-patient interaction.

OBJECTIVE: To evaluate the long-term course and prognosis associated with the irritable bowel syndrome (IBS) and to determine the influence of an effective physician-patient relationship on subsequent health care use. DESIGN: Prospective review of medical records. SETTING: Tertiary referral center. PATIENTS: 112 consecutive Olmsted County, Minnesota, residents who were first diagnosed with IBS at the Mayo Clinic during the period 1961-1963. RESULTS: The median follow-up was 29 years (range, 1 to 32 years) and patients made a median of 2 return visits for IBS-related symptoms (range, 0 to 12 visits). In addition to abdominal pain, diarrhea (reported by 50% of patients) was the predominant bowel symptom at diagnosis. Organic gastrointestinal disease occurred in 10 patients a median of 15 years after diagnosis of IBS. Survival in patients with IBS did not differ from expected survival (27 deaths; median survival > 30 years after initial diagnosis). A positive physician-patient interaction, defined a priori using objective criteria in the written record, was associated with fewer return visits for IBS. Of the eight variables examined, notations in the medical record about psychosocial history, precipitating factors, and discussion of diagnosis and treatment with patients were associated with fewer return visits for IBS-related symptoms. CONCLUSIONS: When diagnosed according to current criteria, IBS is associated with a good prognosis and the diagnosis is unlikely to be changed to that of an organic disease during follow-up. A positive physician-patient interaction may be related to reduced use of ambulatory health services by patients with IBS.