Karyometric analysis of actinic damage in unexposed and sun-exposed skin and in actinic keratoses in untreated individuals

OBJECTIVE: To assess the ability of karyometric analysis to demonstrate progression of actinic damage as a function of sun exposure in individuals with actinic keratoses (AKs) and to evaluate the stability of that assessment over a 3-month period. STUDY DESIGN: Biopsies from subjects with AKs were obtained from unexposed skin, sun-exposed skin and AK lesions. Subjects used an SPF 50 sunscreen, and 3 months later additional biopsies were taken from sun-exposed and AK sites. A total of 13,300 nuclei were recorded from 31 subjects. RESULTS: Measures of nuclear abnormality (NA) and effects of sun damage based on discriminant function (DF) scores were derived. Actinic damage levels varied significantly across biopsy site, demonstrating the method's sensitivity. Accrual of actinic damage was demonstrated in sun-exposed skin and AK lesions when all nuclei were examined over 3 months but only for sun-exposed skin when the worst-damaged nuclei were examined. This suggests a ceiling effect of nuclear damage in the progression to abnormality. Within-subject variability was similar for both NA and DF when all nuclei were considered. Among the worst-damaged nuclei (as defined by high DF), DF showed lower within-case variability than NA, perhaps due to a reduction in nuclear heterogeneity. CONCLUSION: Karyometry's ability to detect subtle levels of actinic damage in nuclear chromatin patterns may make it useful in screening agents for possible use in cancer chemoprevention.     

Measurement of chemopreventive efficacy in skin biopsies

OBJECTIVE: To explore methods suitable for quantitative assessment of the efficacy of chemopreventive intervention. STUDY DESIGN: High-resolution imagery of nuclei from the suprabasal and basal cell layers of sun-damaged skin were recorded. There were 10 cases. A shave biopsy was taken from an area of clearly evident solar keratosis before and after treatment with 2-difluoromethyl-dlornithine (DFMO) and from the colateral forearm, treated with a placebo. A number of karyometric variables were computed and combined to derive marker features that provided a numeric measure of the degree of nuclear deviation from normal. RESULTS: DFMO treatment was effective overall in reducing the degree of nuclear abnormality seen in the biopsies; in 8 of the 10 cases there was a significant improvement. The placebo-treated arm did not show a statistically different abnormality from the untreated arm. CONCLUSION: Karyometric analysis can provide numeric measures that allow documentation of statistically significant regression of actinic keratotic lesions following treatment with DFMO.     

BRAF and RKIP are significantly decreased in cutaneous squamous cell carcinoma

Background. Actinic keratosis (AK) is a well-established pre-cancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). However, little is known about the implication of BRAF and RKIP expression, or about the incidence of BRAF mutations in the formation of these cutaneous diseases. The RAS oncogene has been proposed to significantly contribute to skin cancer development. Moreover, numerous BRAF mutations have been detected in melanoma biopsy specimens and cell lines.

Objectives. This study aimed to measure the mRNA levels of the genes BRAF and RKIP in AK, as well as their possible implication in the progress of AK to SCC. All biopsy specimens were also screened for BRAF mutations within exons 11 and 15.

Patients and methods. Expression levels of the genes BRAF and RKIP were examined in 16 AKs and 12 SCCs by RT-qPCR. A novel allele-specific qPCR method, in combination with direct DNA sequencing, was performed in order to inspect the frequency of the V600E mutation in exon 15, as well as to examine the mutation status of the gene within exon 11.

Results. Significant down-regulation was noted for both genes in SCC, compared to normal tissue (for BRAF, P=0.002; for RKIP, P     

Unbiased Approach for Virus Detection in Skin Lesions

To assess presence of virus DNA in skin lesions, swab samples from 82 squamous cell carcinomas of the skin (SCCs), 60 actinic keratoses (AKs), paraffin-embedded biopsies from 28 SCCs and 72 kerathoacanthomas (KAs) and fresh-frozen biopsies from 92 KAs, 85 SCCs and 92 AKs were analyzed by high throughput sequencing (HTS) using 454 or Ion Torrent technology. We found total of 4,284 viral reads, out of which 4,168 were Human Papillomavirus (HPV)-related, belonging to 15 known (HPV8, HPV12, HPV20, HPV36, HPV38, HPV45, HPV57, HPV59, HPV104, HPV105, HPV107, HPV109, HPV124, HPV138, HPV147), four previously described putative (HPV 915 F 06 007 FD1, FA73, FA101, SE42) and two putatively new HPV types (SE46, SE47). SE42 was cloned, sequenced, designated as HPV155 and found to have 76% similarity to the most closely related known HPV type. In conclusion, an unbiased approach for viral DNA detection in skin tumors has found that, although some new putative HPVs were found, known HPV types constituted most of the viral DNA.     

Statistical analysis of chemopreventive efficacy of vitamin A in sun-exposed,normal skin

OBJECTIVE: To develop numeric, statistically secured measures of chemopreventive efficacy and to derive procedures with high sensitivity of detection. STUDY DESIGN: Karyometric features were computed for nuclei from the basal cell layer of biopsies taken from sun-exposed but histologically "normal" skin. Biopsies were collected from placebo-treated subjects and subjects treated for one year with daily, oral doses of 25,000, 50,000 and 75,000 IU of vitamin A. A total of 22,600 nuclei were recorded from 113 cases, at baseline and after one year. RESULTS: Two numeric measures of chemopreventive efficacy were applied: a measure of nuclear abnormality and a measure based on discriminant function scores. Both showed statistically significant chemopreventive effects of vitamin A. Dose-response curves were derived. A novel procedure, second order discriminant analysis, resulted in very high sensitivity for the detection of change in nuclear chromatin patterns. CONCLUSION: Karyometric analysis has increased in sensitivity such that changes on the order of 10%, found in only a low percentage of nuclei in a biopsy specimen, can be reliably documented. The methodology lends itself to cost-efficient screening of compounds for chemopreventive efficacy.     

Different expression of cyclooxygenase-2 (COX-2) in selected nonmelanocytic human cutaneous lesions

The aim of our study was to elucidate the possible involvement of COX-2 in the development and/or progression of nonmelanocytic skin lesions. To evaluate the usefulness of that enzyme as a potential molecular marker, we examined the intensity and spatial distribution of COX-2 expression in selected types of such tumors using the same immunohistochemical procedure as in our earlier studies of melanocytic cancers. We examined 20 benign epithelial lesions, 11 precancerous lesions, 21 basal cell carcinomas (BCC), 14 squamous cell carcinomas (SCC) and eight fibromas. The levels of COX-2 expression detected in benign lesions and in normal skin were comparable. Elevated expression of this protein may play a role in the development of SCC, as indicated by strong immunostaining both in SCCs and precancerous lesions. Significantly stronger staining in SCCs compared to BCCs may indicate a role of COX-2 in cancer malignancy and serve as an indicator useful for differential diagnostics of the two types of cancer. Strong staining in all skin layers of SCC may help in detecting cancer cells infiltrating surrounding skin layers.     

Reliability and validity of a histologic score as a marker for skin cancer chemoprevention studies

OBJECTIVE: To develop a reliable and valid scoring system for grading skin biopsies from actinic keratosis (AK) and sun-damaged skin for use in evaluating the efficacy of skin cancer chemopreventive agents. STUDY DESIGN: A panel of dermatopathologists developed histologic criteria and diagnostic definitions for the progression of lesions from early AK to AK. The criteria were then applied to a sample of 335 histologic slides from an ongoing chemoprevention study. A 10% sample of 35 slides was reread in order to assess intrarater reliability. RESULTS: Six of the 7 criteria demonstrated high reliability (> 85%). The total histologic score, calculated using the 6 criteria, was found to significantly differentiate between (blinded) biopsy location (normal, pre-AK, AK and adjacent to squamous cell carcinoma) and histologic diagnosis (normal, pre- or early AK, AK and squamous cell carcinoma). CONCLUSION: The total histologic score, having demonstrated reliability on repeated readings and validity in its association with biopsy location and histologic score, is a reliable and valid end point for judging the efficacy of agents in skin cancer chemoprevention studies. Additional interrater reliability tests utilizing larger test sets and a rigorous statistical design should be undertaken to establish its portability.     

Osteopontin expression in normal skin and non-melanoma skin tumors.

Osteopontin (OPN) is an adhesive, matricellular glycoprotein, whose expression is elevated in many types of cancer and has been shown to facilitate tumorigenesis in vivo. To understand the role of OPN in human skin cancer, this study is designed to determine whether OPN is expressed in premalignant [solar/actinic keratosis (AK)] and in malignant skin lesions such as squamous cell carcinomas (SCC) and basal cell carcinomas (BCC), as well as in normal skin exposed or not exposed to sunlight. Immunohistochemical analyses showed that OPN is expressed in SCC (20/20 cases) and in AK (16/16 cases), which are precursors to SCC, but is absent or minimally expressed in solid BCC (17 cases). However, positive staining for OPN was observed in those BCC that manifest differentiation toward epidermal appendages such as keratotic BCC. In sunlight-exposed normal skin, OPN is minimally expressed in the basal cell layer, but in contrast to those not exposed to sunlight, OPN is more prominent in the spinous cell layer with increasing intensity toward the granular cell layer. Additionally, OPN is expressed in the hair follicles, sebaceous glands, and sweat glands of normal skin. In conclusion, these data suggest that OPN is associated with keratinocyte differentiation and that it is expressed in AK and SCC, which have metastatic potential, but minimally expressed in solid BCC.     

Actinic keratosis associated with squamous and basal cell carcinomas: an evaluation of neoplastic progression by a standardized AgNOR analysis

In an attempt to investigate the neoplastic progression in different stages of actinic keratosis (AK), a standardized AgNOR analysis was performed in 94 cases of AK, 35 of which were associated with squamous cell carcinoma (SCC) or basal cell carcinoma (BCC), and in 31 cases of SCC and 22 cases of BCC. The cases were subdivided into low- and high-AgNOR-expressing (AgNOR status) AK by using the mean area of AgNORs per cell (NORA) value (3.996 micro(2)) as the cut-off. In AK samples, a progressive increase of the mean NORA value from Stage I to Stage IV was encountered. In addition, a significantly higher mean NORA value was found in the AK cases associated with SCC, in comparison to those without SCC; by contrast, no significant differences in the mean NORA value were noted between AK cases with or without BCC. A highly significant association between a high AgNOR quantity and the coexistence of SCC was encountered in AK; no association was appreciable between the AgNOR quantity and the co-occurrence of BCC. Moreover, when the co-existence of SCC in AK was considered as the reference point, the AK cases associated with SCC mostly (95.5%) presented a high AgNOR quantity (high sensitivity), but only 57.6% of cases without SCC displayed a low AgNOR quantity (low specificity). Additionally, our data document that the standardised AgNOR analysis represents a strong negative predictor for the association between SCC and AK. Indeed, a low AgNOR quantity mostly is associated with AK cases without SCC.     

Non‐invasive monitoring of subclinical and clinical actinic keratosis of face and scalp under topical treatment with ingenol mebutate gel 150 mcg/g by means of reflectance confocal microscopy and optical coherence tomography: New perspectives and comparison of diagnostic techniques

Actinic keratosis (AK) corresponds to the earliest stage of in situ squamous cell carcinoma and arises on chronically sun‐exposed skin. Around the clinically evident AKs, the apparently healthy epidermis may contain different grades of atypia that can be detected by noninvasive imaging techniques such as reflectance confocal microscopy (RCM) and optical coherence tomography (OCT). Subclinical actinic keratosis (sAK) has captured increasing interest as a potential target of field therapies. The aim of this study was to evaluate in vivo the changes in the field cancerization undergoing treatment with topical ingenol mebutate by combining RCM and OCT. Twenty patients with field cancerization of the face and scalp were treated with ingenol mebutate gel (150 mcg/g) for three consecutive days on an area of 25 cm² containing at least two AKs, two sAKs and two apparently healthy sites. About 120 lesions were evaluated through clinical investigation and clinical, dermoscopical, RCM and OCT images at day 0, 4, 14 and 56 based on the diagnostic criteria for AKs. Main pathological features improved in both AKs and sAKs, in particular the epidermal thickness measured by OCT and the epidermal atypia graded by RCM. Local skin reactions (LSR) arose predominantly in the lesional area compared with healthy skin. A complete clearance was detected in 58% for AKs, and in 55% and 72% for sAKs measured by RCM and OCT, respectively. Both OCT and RCM allow the morphological representation of field cancerization including subclinical lesions and provide complementary information. Ingenol mebutate is effective not only in clinically evident but also in sAKs. The differences in LSR highlight the potential selectivity of the treatment.      

The multi-step process of human skin carcinogenesis: a role for p53, cyclin D1, hTERT, p16, and TSP-1

As proposed by Hanahan and Weinberg (2000. Cell 100, 57-70) carcinogenesis requires crucial events such as (i) genomic instability, (ii) cell cycle deregulation, (iii) induction of a telomere length maintenance mechanism, and (iv) an angiogenic switch. By comparing the expression of p53, cyclin D1, p16, hTERT, and TSP-1 in spontaneously regressing keratoacanthoma (KA) as a paradigm of early neoplasia, with malignant invasive cutaneous squamous cell carcinoma (SCC) as a paradigm of advanced tumour development, we are now able to assign the changes in the expression of these proteins to specific stages and allocate them to defined roles in the multi-step process of skin carcinogenesis. We show that mutational inactivation of the p53 gene, and with that the onset of genomic instability is the earliest event. Individual p53-positive cells are already seen in "normal" skin, and 3/5 actinic keratoses (AKs), 5/22 KAs, and 13/23 SCCs contain p53-positive patches. Cell cycle deregulation was indicated by the overexpression of the cell cycle regulator cyclin D1, as well as by the loss of the cell cycle inhibitor p16. Interestingly, overexpression of cyclin D1 - observed in 80% of KAs and SCCs, respectively - showed a cell cycle-independent function in HaCaT cell transplants on nude mice. Cyclin D1 overexpression was associated with a massive inflammatory response, finally leading to tissue destruction. Loss of the cell cycle inhibitor p16, on the other hand, correlated with SCCs. Thus, it is tempting to suggest that overexpression of cyclin D1 is an early change that in addition to growth stimulation leads to an altered epithelial-mesenchymal interaction, while functional p16 is able to control this deregulated growth and needs to be eliminated for malignant progression. Another requirement for uncontrolled growth is the inhibition of telomere erosion by up-regulating telomerase activity. As measured by hTERT protein expression, all of the KAs and SCCs studied were positive, with a similar distribution of the protein in both groups and an expression pattern resembling that of normal epidermis. Thus, telomerase may not need to be increased significantly in skin carcinomas. Finally, we show that the angiogenesis inhibitor TSP-1 is strongly expressed in most KAs, and mainly by the tumour cells, while in SCCs the generally weak expression is restricted to the tumour-stroma. Furthermore, we provide evidence that the loss of a copy of chromosome 15 is responsible for reduced TSP-1 expression and thereby this aberration contributes to tumour vascularisation (i.e. the angiogenic switch) required for malignant growth.     

Karyometry in Barrett's esophagus

OBJECTIVE: To derive a progression curve for lesions in Barrett's esophagus based on karyometric features. STUDY DESIGN: High-resolution imagery of 900 nuclei from normal gastric tissue, Barrett's metaplasia, Barrett's high grade dysplasia and adenocarcinoma of the esophagus was recorded. Karyometric features were computed, and nuclear signatures and lesion signatures for these lesions were derived. A progression curve was defined. RESULTS: Esophageal lesions were distinctly different from the normal gastric fundus tissue, with nuclei from Barrett's metaplasia deviating from normal almost as much as nuclei from high grade dysplasia and adenocarcinoma. There was considerable case-to-case variability and overlap between lesions histologically assigned to different diagnostic categories. CONCLUSION: The karyometric data suggest that Barrett's metaplasia is a more developed lesion than previously assumed.     

The Early Evolution of the Phosphagen Kinases—Insights from Choanoflagellate and Poriferan Arginine Kinases

Arginine kinase (AK) is a member of a large family of phosphoryl transfer enzymes called phosphagen (guanidino) kinases. AKs are present in certain protozoans, sponges, cnidarians, and both lophotrochozoan and ecdysozoan protostomes. Another phosphagen kinase, creatine kinase (CK), is found in sponges, cnidarians, and both deuterostome and protostome groups but does not appear to be present in protozoans. To probe the early evolution of phosphagen kinases, we have amplified the cDNAs for AKs from three choanoflagellates and from the hexactinellid sponge Aphrocallistes beatrix and the demosponges Suberites fuscus and Microciona prolifera. Phylogenetic analysis using maximum likelihood of these choanoflagellate and sponge AKs with other AK sequences revealed that the AK from the choanoflagellate Monosiga brevicollis clusters with the AK from the glass sponge Aphrocallistes and is part of a larger cluster containing AKs from the demosponges Suberites and Microciona as well as basal and protostome invertebrates. In contrast, AKs from Codonosiga gracilis and Monosiga ovata form a distinct cluster apart from all other AK sequences. tBLASTn searches of the recently released M. brevicollis genome database showed that this species has three unique AK genes—one virtually identical to the M. brevicollis cDNA and the other two showing great similarity to C. gracilis and M. ovata AKs. Three distinct AK genes are likely present in choanoflagellates. Two of these AKs display extensive similarity to both CKs and an AK from sponges. Previous work has shown CK evolved from an AK-like ancestor prior to the divergence of sponges. The present results provide evidence suggesting that the initial gene duplication event(s) leading to the CK lineage may have occurred before the divergence of the choanoflagellate and animal lineages.     

Genetic tumor archeology: microdissection and genetic heterogeneity in squamous and basal cell carcinoma

Carcinogenesis is a multi-step series of somatic genetic events. The complexity of this multi-hit process makes it difficult to determine each single event and the definitive outcome of such events. To investigate the genetic alterations in cancer-related genes, sensitive and reliable detection methods are of major importance for generating relevant results. Another critical issue is the quality of starting material which largely affects the outcome of the analysis. Microdissection of cells defined under the microscope ensures a selection of representative material for subsequent genetic analysis. Skin cancer provides an advantageous model for studying the development of cancer. Detectable lesions occur early during tumor progression, facilitating molecular analysis of the cell populations from both preneoplastic and neoplastic lesions. Alterations of the p53 tumor suppressor gene are very common in non-melanoma skin cancer, and dysregulation of p53 pathways appear to be an early event in the tumor development. A high frequency of epidermal p53 clones has been detected in chronically sun-exposed skin. The abundance of clones containing p53 mutated keratinocytes adjacent to basal cell (BCC) and squamous cell carcinoma (SCC) suggests a role in human skin carcinogenesis. Studies using p53 mutations as a clonality marker have suggested a direct link between actinic keratosis, SCC in situ and invasive SCC. Microdissection-based studies have also shown that different parts of individual BCC tumors can share a common p53 mutation yet differ with respect to additional alterations within the p53 gene, consistent with subclonal development within tumors. Here, we present examples of using well-defined cell populations, including single cells, from complex tissue in combination with molecular tools to reveal features involved in skin carcinogenesis.     

Cytological diagnosis of basal cell carcinoma and actinic keratosis, using Papanicolaou and May–Grünwald–Giemsa stained cutaneous tissue smear

Objective:  Cytology may become the diagnostic method of choice with the advent of new non-invasive treatments for non-melanoma skin cancer, as the sampling technique for cytology entails little tissue disfiguration. The aim of this study was to compare and evaluate the diagnostic performance of scrape cytology using two different cytological staining techniques, and to evaluate additional touch imprint cytology, with that of histopathology of basal cell carcinoma (BCC) and actinic keratosis (AK).
Methods:  We investigated 50 BCC and 28 AK histologically verified lesions, from 41 and 25 patients, respectively. Two separate skin scrape samples and one touch imprint sample were taken from each lesion. The smears were stained with Papanicolaou (Pap) or May–Grünwald–Giemsa (MGG) stains. All cytological specimens were examined in random order by pathologists without knowledge of the histology. Cytodiagnostic results were compared with the histopathological report.
Results:  Scrape cytodiagnosis agreed with histopathology in 48 (Pap) and 47 (MGG) of the 50 BCC cases, and in 26 of 28 (Pap) and 21 of 26 (MGG) AK cases, yielding sensitivities of 96%, 94%, 93% and 81%, respectively. No significant difference in sensitivity between the two staining methods was found but a trend towards higher Pap sensitivity for AK was noted ( P  =   0.10). Touch imprint cytology confirmed histopathology in 38 of the 77 cases of BCC and AK.
Conclusion:  Cytological diagnosis with either Pap or MGG stain for BCC and AK is reliable, and differentiates well between BCC and AK. Imprint cytology proved to be non-diagnostic in half of the examined cases.     

Physiological model using diffuse reflectance spectroscopy for nonmelanoma skin cancer diagnosis

Diffuse reflectance spectroscopy (DRS) is a noninvasive, fast, and low‐cost technology with potential to assist cancer diagnosis. The goal of this study was to test the capability of our physiological model, a computational Monte Carlo lookup table inverse model, for nonmelanoma skin cancer diagnosis. We applied this model on a clinical DRS dataset to extract scattering parameters, blood volume fraction, oxygen saturation and vessel radius. We found that the model was able to capture physiological information relevant to skin cancer. We used the extracted parameters to classify (basal cell carcinoma [BCC], squamous cell carcinoma [SCC]) vs actinic keratosis (AK) and (BCC, SCC, AK) vs normal. The area under the receiver operating characteristic curve achieved by the classifiers trained on the parameters extracted using the physiological model is comparable to that of classifiers trained on features extracted via Principal Component Analysis. Our findings suggest that DRS can reveal physiologic characteristics of skin and this physiologic model offers greater flexibility for diagnosing skin cancer than a pure statistical analysis. Physiological parameters extracted from diffuse reflectance spectra data for nonmelanoma skin cancer diagnosis.     

Arginine kinase from the Tardigrade, Macrobiotus occidentalis: molecular cloning, phylogenetic analysis and enzymatic properties

Arginine kinase (AK), which catalyzes the reversible transfer of phosphate from ATP to arginine to yield phosphoarginine and ADP, is widely distributed throughout the invertebrates. We determined the cDNA sequence of AK from the tardigrade (water bear) Macrobiotus occidentalis, cloned the sequence into pET30b plasmid, and expressed it in Escherichia coli as a 6x His-tag—fused protein. The cDNA is 1377 bp, has an open reading frame of 1080 bp, and has 5′- and 3′-untranslated regions of 116 and 297 bp, respectively. The open reading frame encodes a 359-amino acid protein containing the 12 residues considered necessary for substrate binding in Limulus AK. This is the first AK sequence from a tardigrade. From fragmented and non-annotated sequences available from DNA databases, we assembled 46 complete AK sequences: 26 from arthropods (including 19 from Insecta), 11 from nematodes, 4 from mollusks, 2 from cnidarians and 2 from onychophorans. No onychophoran sequences have been reported previously. The phylogenetic trees of 104 AKs indicated clearly that Macrobiotus AK (from the phylum Tardigrada) shows close affinity with Epiperipatus and Euperipatoides AKs (from the phylum Onychophora), and therefore forms a sister group with the arthropod AKs. Recombinant 6x His-tagged Macrobiotus AK was successfully expressed as a soluble protein, and the kinetic constants (K(m), K(d), V(ma) and k(cat)) were determined for the forward reaction. Comparison of these kinetic constants with those of AKs from other sources (arthropods, mollusks and nematodes) indicated that Macrobiotus AK is unique in that it has the highest values for k(cat) and K(d)K(m) (indicative of synergistic substrate binding) of all characterized AKs.     

Arginine kinase from the beetle Cissites cephalotes (Olivier). Molecular cloning, phylogenetic analysis and enzymatic properties

Here, we report the PCR amplification and cloning of a cDNA for arginine kinase (AK) from the beetle Cissites cephalotes (Olivier). The cDNA is 1210bp and has an open reading frame of 1125bp and 5' and 3'-untranslated regions of 30 and 55bp, respectively. The open reading frame encodes a 374 amino acid protein with most of the residues considered necessary for AK function: five residues predicted to interact with the substrate arginine (S77, Y82, E239, C285 and E328), and five residues predicted to interact with the substrate ADP (R138, R140, R243, R294 and R323). A phylogenetic tree of arthropod AKs indicated clearly that insect AKs can be separated into typical AKs from various insect species (group 1) and putative AK sequences deduced from genomic sequences (group 2). Cissites AK clustered in group 2 and provides the first evidence that a group-2 gene is indeed expressed in insects. Moreover, we expressed Cissites AK protein in Escherichia coli as a fusion with maltose-binding protein, and kinetic constants (K(m), K(d), V(max) and k(cat)) were determined for the forward reaction. Comparison of kinetic constants with those of AKs from other sources (insects, mollusks and echinoderms) indicated that insect AKs from Cissites and Periplaneta have two very unique features, the lowest k(cat) (and k(cat)/K(m)(arg)) among AKs, and a lack of synergistic substrate binding (K(d)/K(m) approximately 1).     

Significance of eIF4E expression in skin squamous cell carcinoma

Cutaneous squamous cell carcinoma (SCC) is a malignant tumour of keratinising epidermal cells. This type of skin cancer is the second leading cause of death after melanoma, and it is the second most common type of non-melanoma skin cancer after basal cell carcinoma. The cellular and molecular events involved in the progression of skin cancers are largely unknown. Increased protein synthesis is necessary for the transition of cells from quiescence to proliferation. Translational control is critical for the proper regulation of the cell cycle, tissue induction and growth. Eukaryotic initiation factor eIF4E, an important regulator of translation, plays critical roles in neo-plastic transformation and cancer progression. We investigated eIF4E expression in 49 skin samples (six normal tissues, eight Bowen diseases, seven stage I, 10 stage II, 13 stage III and five stage IV SCCs). Results obtained demonstrated that all SCC samples, evaluated by SDS-PAGE, Western blotting and cap-affinity chromatography using m7GTP-sepharose, presented eIF4E expression (13.6+/-1.2), whereas, starting from stage 0 (4.1+/-0.9) to stage I (7.4+/-1.4), stage II (12.1+/-2.4), stage III (18.1+/-3.0) and stage IV (26.2+/-3.8) SCCs, a constant and significant increase of protein over expression (P<0.001) was observed. A high expression of eIF4E is correlated with advanced stages. The results presented in this study demonstrate a possible role of eIF4E in SCC.