Impaired hair follicle morphogenesis and cycling with abnormal epidermal differentiation in nackt mice,a cathepsin L-deficient mutation

We previously described an autosomal-recessive mutation named nackt (nkt) exhibiting partial alopecia associated with CD4(+) T-cell deficiency. Also, we recently reported that nkt (now Ctsl(nkt)) comprises a deletion in the cathepsin L (Ctsl) gene. Another recent study reported that Ctsl knockout mice have CD4(+) T-cell deficiency and periodic shedding of hair, which recapitulate the nkt mutation and the old furless (fs) mutation. The current study focuses on the dermatological aspects of the nkt mutation. Careful histological analysis of skin development of homozygous nkt mice revealed a delayed hair follicle morphogenesis and late onset of the first catagen stage. The skin of Ctsl(nkt)/Ctsl(nkt) mice showed mild epidermal hyperplasia and hyperkeratosis, severe hyperplasia of the sebaceous glands, and structural alterations of hair follicles. Epidermal differentiation seems to be affected in nkt skin, with overexpression of involucrin and profilaggrin/filaggrin along with focal areas of keratin 6 expression in the interfollicular epidermis. Severe epidermal hyperplasia, acanthosis, orthokeratosis, and hyperkeratosis were only observed in mice maintained in nonpathogen-free environments. The analysis of Rag2-/- Ctsl(nkt)/Ctsl(nkt) double-mutant mice indicates that the skin defect remains under the absence of T and B cells. This animal model provides in vivo evidence that cysteine protease cathepsin L plays a critical role in hair follicle morphogenesis and cycling, as well as epidermal differentiation.     

Angiogenesis is an early event in the development of chemically induced skin tumors

In this study we have analyzed the vascular response induced in the two- stage carcinogenesis model in SENCAR mice. The role of angiogenesis has not been explored in this model, which is the paradigm of multistage carcinogenesis and a model for neoplastic lesions derived from exophytic premalignant lesions (e.g. colon carcinoma, bladder papilloma). We investigated if angiogenesis is involved in the formation of papillomas and in the progression from papilloma to carcinoma. To this end we analyzed the vasculature of normal and hyperplastic skin, focal epidermal hyperplasias that are precursors of papillomas, papillomas at different stages and squamous cell carcinomas. We also analyzed the vascularization of papillomas induced in two strains of mice that differ in their susceptibility to malignant progression. We show here that angiogenesis is turned on in the earliest stages of papilloma formation. In late stages, regardless of state of progression, the predominant response is an increase in the size of blood vessels. Thus, in the SENCAR mouse model, representative of exophytic tumors, the angiogenesis switch is a very early event, probably mechanistically related to the development of the primarily exophytic lesions. Therefore, the density of blood vessels cannot be used as a predictor of malignant progression in this model.      

p53 alterations in chemically induced hamster cheek-pouch lesions

To confirm that the hamster cheek-pouch carcinogenesis model reflects development of human squamous cell carcinoma (SCC), we determined if and when p53 mutations occur in the development of SCC in this model by using immunohistochemical staining and polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis plus direct DNA sequencing. Twenty-four hamster cheek-pouches were treated with a solution of 0.5% 7,12-dimethylbenz[a]anthracene in mineral oil three times a week for 16 wk. The malignant endophytic and exophytic tumors induced with this protocol are preceded by a sequence of premalignant lesions such as hyperplasia with or without dysplasia and carcinoma in situ, similar to the development of this cancer in humans. For this study, p53 protein accumulation was evaluated by immunostaining of various hamster cheek-pouch exophytic and endophytic SCCs as well as flat dysplastic hyperplasia and carcinomas in situ. A moderate percentage (33.3%) of exophytic lesions and most endophytic carcinomas (90%) showed positive p53 staining. In addition we also found p53-positive staining in a number of preneoplastic lesions, including areas of focal hyperplasia, dysplastic hyperplasia, and carcinomas in situ. To determine whether the alterations in p53 staining were due to p53 gene mutation, we used PCR-SSCP analysis and direct sequencing. PCR products corresponding to exons 5a, 6, 7, and 8 from 40 tumors with the highest percentage of p53-stained cells were analyzed. We detected shifted bands in 17 lesions. Direct sequencing of eight selected shifted bands revealed four mutations, including two G→T transversions in codons 216 (tumor #1) and 252 (tumor #2) and one G→C transversion in codon 282 (tumor #3). Tumor #4 contained a frameshift mutation in codon 251. These mutations are consistent with those reported in many human cancers. Therefore, we concluded that in the hamster cheek-pouch model, p53 protein accumulation occurs frequently and early in carcinogenesis, as it does in human SCCs, and some of these p53 alterations are due to p53 gene mutations. These findings may help us better define the mechanisms of carcinogenesis in the hamster cheek-pouch model, and p53 alterations may be an early biomarker of progression for chemoprevention studies. © 1996 Wiley-Liss, Inc.     

Prognostic significance of urokinase plasminogen activator receptor and its cleaved forms in blood from patients with non‐small cell lung cancer

Urokinase plasminogen activator (uPA) cleaves its three‐domain cell surface receptor, uPAR, liberating domain I [uPAR(I)] and leaving the cleaved uPAR(II‐III) on the cell surface. Both intact and cleaved uPAR can be shed from the cell surface. uPAR(I) was previously shown to be a prognostic factor in lung tumour extracts. Here we analyse uPAR forms in blood from patients with non‐small cell lung cancer (NSCLC). Preoperatively sampled plasma/serum from 32 patients with NSCLC was analysed. Three time‐resolved fluoroimmunoassays (TR‐FIAs) measuring intact uPAR(I‐III) (TR‐FIA 1), uPAR(I‐III) + uPAR(II‐III) (TR‐FIA 2) and uPAR(I) (TR‐FIA 3) were applied. The Spearman rank correlations between plasma and serum levels of uPAR(I‐III), uPAR(I‐III) + uPAR(II‐III), and uPAR(I) were 0.89, 0.94 and 0.68 respectively. Survival analysis demonstrated that high levels of all uPAR forms were associated with shorter survival. Adjusted for histological subtype high plasma uPAR(I‐III) and uPAR(I) levels as well as serum uPAR(I) levels were significantly associated with shorter OS (hazards ratios = 4.3, 2.8 and 3.8 respectively). High blood levels of intact uPAR and its cleaved forms are associated with poor prognosis in NSCLC.     

ACCELERATED PAPER: Intermittent 50 Hz magnetic field and skin tumour promotion in SENCAR mice

A number of epidemiological studies have indicated associationbetween exposure to extremely low frequency electromagneticfields and a variety of cancers, including leukaemia and braintumours among residentially exposed children and among occupationallyexposed adults. In order to test if intermittent magnetic fields(MF) act as a tumour promoter, a long-term skin carcinogenicitystudy of 50 Hz sinusoidal MF with flux densities of 50 µTand 0.5 mT, continuous as well as with an intermittence of 15s on/off, was performed. Female SENCAR mice were divided intoeight groups of 50 animals in each and treated according toan initiation- promotion scheme. 7,12-dimethylbenz[a] anthracene(DMBA) in acetone was applied to the dorsal skin at a subcarcinogenicdose, as an initiator and exposure to MF was performed for 19–21h/day during 104 weeks starting 1 week after the initiator treatment.The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)was used as a positive control for skin tumour promoting activity.Two animals from each group were assigned for skin hyperplasiaanalysis at 2, 6, 12,18 and 21 months. The animals were observeddaily. The appearance of skin lesions and neoplasms were carefullyfollowed and histopathological diagnosis was made for all neoplasmspresent at death. The experiment was terminated after 105 weeks.DMBA-treatment alone yielded altogether two skin tumours intwo tumour-bearing animals and the animals exposed to acetonealone had one skin tumour. The animals exposed to continuousfields showed no skin tumour. Five animals exposed to 0.5 µTon/off had a total of 13 skin tumours and in the group exposedto 50 µT on/off four animals had a total of four skintumours. The on/off exposed groups differed significantly fromthe continuously exposed groups (P = 0.014) but the differencebetween the on/off exposure groups and the DMBA group was notstatistically significant when tumour-bearing animals and cumulatedskin tumours were compared. There was a statistically significantdose trend (P = 0.045) with flux density and Tesla-h for intermittentMF exposure for cumulated skin tumours per tumour-bearing animals.The epithelial thickness of DMBA + MF-treated animals was ofthe same magnitude as for DMBA-treated animals indicating that,in the case of a promoting effect being present, another mechanismthan one involving sustained hyperplasia may be involved.     

A study on skin tumour formation in mice with 50 Hz magnetic field exposure

In order to test the possibility that magnetic fields (MF) actas a tumour promoter, a long-term skin carcinogenicity studyof 50 Hz sinusoidal MF with flux densities of 50 µT and0.5 mT was performed in female NMRI mice. 7, 12–dimethylbenz[a]anthracene(DMBA) in acetone was applied to the dorsal skin, as an initiator,and exposure to MF was performed for 19 (weekdays) or 21 h/day(weekends and holidays) for 103 weeks starting one week afterthe initiator treatment. The phorbol ester 12–O–tetradecanoylphorbol-13-acetate(TPA) was used as a positive control for skin tumour promotingactivity. MF was also evaluated for complete carcinogenic actionin groups of mice that were treated with acetone only. Six animalsfrom each group were taken for skin hyperplasia analysis andwere killed after 9, 26 and 52 weeks. The appearance of skinlesions were carefully followed and histopathological diagnosiswas made for all neoplasms present at death. The statisticalanalyses on occurrence of skin tumour bearing animals and cumulatedskin tumours, with corrections for survival, did not reveala difference between the controls and the MF exposed groups.The epithelial thickness of DMBA + MF-treated animals was ofthe same magnitude as for DMBA-treated animals. Leukaemia wasa little more frequent among animals exposed to 0.5 mT MF comparedto the control animals. However this difference was not statisticallysignificant.     

Low frequency of codon 61 Ha-ras mutations and lack of keratin 13 expression in 7,12-dimethylbenz[a]-anthracene—induced hamster skin tumors

Alterations in the pattern of keratin expression are a common feature of skin-tumor development. In this study, we investigated whether the loss of epidermal keratin 1 (K1) and its replacement by mucosal keratin 13 (K 13) is unique to mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), since it has been reported that human epidermal tumors do not exhibit aberrant expression of K13. With that purpose, we analyzed the keratin profiles of 16 DMBA-induced hamster skin tumors using monospecific antibodies against K1 and K13. Although all the tumors expressed K1, they also showed an overall tendency towards loss of this keratin; furthermore, none of the tumors expressed K13. Previous studies have suggested that the induction of K13 in mouse skin is related to the mutation of the Ha-ras gene by the initiating agent DMBA, a mutation consistently found in murine DMBA/TPA-induced tumors and rarely found in human skin tumors. Therefore, we also evaluated the tumors for the presence of codon-61 mutations by direct sequencing of DNA extracted from paraffin-embedded tissue sections. Only three tumors showed an A→T transversion in the second nucleotide of Ha-ras codon 61. However, presence of the mutation did not correlate with K1 staining. Although hamster skin tumors were induced by the same initiator as were mouse skin tumors, hamster skin tumors did not show the same keratin profile. Moreover, their immunohistochemical expression of K1 and K13 and their codon 61 sequences resembled that of their human counterparts. These results suggest that the aberrant expression of K13 may be unique to murine skin. Furthermore, although codon 61 Ha-ras mutation appears to be related to keratin alterations in the mouse model, this mutation is not sufficient to produce the same biochemical changes in other species.