Epidermolysis bullosa simplex due to KRT5 mutations: mutation-related differences in cellular fragility and the protective effects of trimethylamine N-oxide in cultured primary keratinocytes.

BACKGROUND: Epidermolysis bullosa simplex (EBS) is a mechanobullous skin fragility disease characterized by cytolysis of basal keratinocytes and intraepidermal blistering often caused by mutations in keratin genes (KRT5 or KRT14). No remedies exist for these disorders presenting a need for development of novel therapies. OBJECTIVES: To identify new genotype-phenotype relationships in vivo and in cultured primary EBS keratinocytes in vitro, and to study the cytoskeletal stabilizing effects of trimethylamine N-oxide (TMAO) in heat-stressed EBS cells. METHODS: Genomic DNA and cDNA samples from three Swedish patients with EBS were analysed for keratin mutations. Primary EBS keratinocyte cultures were established, heat stressed with and without added TMAO, followed by evaluation of cellular fragility. RESULTS: In addition to the previously reported KRT5 mutation (V186L) in one patient, two patients were found to have a novel I183M and recurrent E475G replacements in KRT5. Cultured EBS keratinocytes did not exhibit keratin aggregates or cell loss, except in the patient with the p.I183M mutation who showed 3% aggregates and 2% cell loss. Upon transient heat stress the number of aggregate-containing cells increased to 21%, 27% and 13%, respectively, in the p.I183M, p.E475G and p.V186L mutant cells. Interestingly, pretreatment with TMAO prior to heat stress, dose dependently reduced the number of aggregate-containing cells and cell loss. CONCLUSION: These results revealed a genotype-phenotype correlation in EBS keratinocytes upon heat stress and suggest protein stabilization as a new therapeutic strategy.

Epidermolysis bullosa in calves in the United Kingdom.

Epidermolysis bullosa (EB) was diagnosed in eight calves from four farms in the United Kingdom on the basis of clinical, histological and ultrastructural findings. In three affected herds, pedigree Simmental bulls had been mated with Simmental-cross cows. In a fourth herd two Holstein-Friesian calves were affected. Lesions included multifocal erosion and ulceration of the hard and soft palates, tongue, nares and gingiva, with onychomadesis (dysungulation). There was alopecia, erosion and crusting of the coronets, pasterns, fetlocks, carpi, hocks, flanks and axillae. Histopathological findings included segmental separation of full thickness epidermis from the dermis, with formation of large clefts containing eosinophilic fluid, extravasated red blood cells and small numbers of neutrophils. Follicular and interfollicular areas of skin were affected, with clefts extending around hair follicles and sometimes involving whole follicles. Ultrastructurally, there was evidence of vacuolar change within basal keratinocytes, corresponding to areas of histological clefting. Preliminary genetic screening of the candidate keratin genes (bKRT5 and bKRT14) has excluded mutations of these as the cause of this condition.

Photosensitive Smith-Lemli-Opitz syndrome is not caused by a single gene mutation: analysis of the gene encoding 7-dehydrocholesterol reductase in five U.K. families.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive malformation syndrome characterized by a disorder in cholesterol metabolism. SLOS is caused by mutations in the DHCR7 gene which encodes 7-dehydrocholesterol reductase, an enzyme that catalyses the final step in cholesterol biosynthesis. We have previously established the clinical and photobiological features of the photosensitivity that is frequently a feature of SLOS. OBJECTIVES: In this study, we have performed mutational analysis of the DHCR7 gene in individuals from five families with SLOS. In each family, one member was affected by severe photosensitivity as a manifestation of SLOS. METHODS: Fifteen samples (including family controls) were screened using polymerase chain reaction amplification and direct automated sequencing. RESULTS: Six different DHCR7 mutations were identified of which five were single point mutations that caused missense amino acid substitutions (P51H, T93M, L99P, E448K and R450L). The other was a splice site mutation (G-->C in splice acceptor site) affecting the intron 8-exon 9 splice junction (IVS8-1 G-->C). This splice site mutation and four of the five missense mutations have been previously published as causal in SLOS but the P51H is a novel mutation which has not previously been reported. CONCLUSIONS: This is the first study in which DHCR7 gene mutational analysis has been performed on SLOS subjects with severe photosensitivity and indicates that no single mutation is responsible for the photosensitivity which characterizes this disorder.

A mutation (N177S) in the structurally conserved helix initiation peptide motif of keratin 5 causes a mild EBS phenotype.

Epidermolysis bullosa simplex (EBS) is a group of predominantly autosomal dominant hereditary disorders of the skin, which manifest as superficial skin blisters after minimal mechanical trauma. Three subtypes have been defined, based on clinical severity. Mutations affecting the genes encoding the epidermal keratins 5 (K5) and 14 (K14) have been linked to the disease, and generally those affecting the helix initiation and termination peptide motifs have been linked to severe EBS phenotypes. We report here a novel mutation in the helix initiation peptide of K5, N177S, that causes only a mild EBS-Weber Cockayne phenotype (EBS-WC). The mutation was identified by direct sequencing of polymerase chain reaction (PCR)-amplified genomic DNA encoding the exons of the KRT5 and KRT14 genes, and confirmed by mismatch allele-specific PCR, followed by restriction enzyme digestion with Tsp509 I. The patient is heterozygous for a mutation affecting codon 177, changing a conserved asparagine residue (N) to serine (S). Asparagine 177 is a highly conserved residue among all type II keratins. This is also the first report of a mutation at position 9 of 1A helix (1A:N9S) in a type II keratin. Unlike mutations affecting residues 4, 5, 7, 8, 10, and 11 of the 1A helix of K5 and K14, which were all previously linked to more severe (EBS) phenotypes, K5 1A:N9S produces only a mild EBS-WC phenotype.

Phenotypic/genotypic correlations in patients with epidermolytic hyperkeratosis and the effects of retinoid therapy on keratin expression.

Dominant-negative mutations in the KRT1 and KRT10 genes cause epidermolytic hyperkeratosis, a rare form of ichthyosis sometimes associated with palmoplantar keratoderma. Although there is no permanent cure, some patients improve on retinoid therapy. More knowledge is needed, however, about the mechanism of action of retinoids and the genotypic/phenotypic correlations in this disease. Thirteen patients from 10 families with generalized disease and 2 sporadic patients with nevoid lesions were studied, probably representing most of the patients in Sweden and Norway. All patients, except one nevoid case, were known to have KRT1 or KRT10 mutations. Those with mutated keratin 1 (K1) invariably had associated keratoderma (n=6). In contrast, only 1 of 7 patients with K10 mutations had this problem (p = 0.0047). Five out of 6 patients with KRT10 mutations benefited from treatment with oral acitretin (5-25mg/day) or topical tretinoin/tazarotene, but none of the patients with KRT1 mutations derived any benefit. Quantitative analysis of K1 and K10 mRNA in skin biopsies obtained before and after retinoid therapy (n=8) showed no consistent down-regulation of mutated keratin that would explain the therapeutic outcome. Instead, the mRNA expression of K2e (a normal constituent of the upper epidermis) diminished especially in nonresponders. In contrast, K4 mRNA and protein (marker of retinoid bioactivity in normal epidermis) increased in almost all retinoid-treated patients. In conclusion, our study confirms a strong association between KRT1 mutations and palmoplantar keratoderma. Retinoid therapy is particularly effective in patients with KRT10 mutations possibly because they are less vulnerable to a down-regulation of K2e, potentially functioning as a substitute for the mutated protein in patients with KRT1 mutations.

A novel keratin 5 mutation (K5V186L) in a family with EBS-K: a conservative substitution can lead to development of different disease phenotypes.

Epidermolysis bullosa simplex is a hereditary skin blistering disorder caused by mutations in the KRT5 or KRT14 genes. More than 50 different mutations have been described so far. These, and reports of other keratin gene mutations, have highlighted the existence of mutation "hotspots" in keratin proteins at which sequence changes are most likely to be detrimental to protein function. Pathogenic mutations that occur outside these hotspots are usually associated with less severe disease phenotypes. We describe a novel K5 mutation (V186L) that produces a conservative amino acid change (valine to leucine) at position 18 of the 1A helix. The phenotype of this case is unexpectedly severe for the location of the mutation, which lies outside the consensus helix initiation motif mutation hotspot, and other mutations at this position have been associated in Weber--Cockayne (mild) epidermolysis bullosa simplex only. The mutation was confirmed by mismatch-allele-specific polymerase chain reaction and the entire KRT5 coding region was sequenced, but no other changes were identified. De novo K5/K14 (mutant and wild-type) filament assembly in cultured cells was studied to determine the effect of this mutation on filament polymerization and stability. A computer model of the 1A region of the K5/K14 coiled-coil was generated to visualize the structural impact of this mutation and to compare it with an analogous mutation causing mild disease. The results show a high level of concordance between genetic, cell culture and molecular modeling data, suggesting that even a conservative substitution can cause severe dysfunction in a structural protein, depending on the size and structure of the amino acid involved.

Two different mutations in the same codon of a type II hair keratin (hHb6) in patients with monilethrix.

Monilethrix is an autosomal dominant hair disorder characterized by a beaded appearance of the hair due to periodic thinning of the shaft. The phenotype shows variable penetrance and results in hair fragility and patchy dystrophic alopecia. Mutations of the helix-encoding region in two hair-specific keratins (hHb1 and hHb6) have been identified. We have now investigated two unrelated monilethrix patients and identified two different novel heterozygous point mutations of the same codon in exon 7 of the hHb6 gene. Dystrophic hair samples obtained from both patients showed the typical beaded appearance by scanning electron microscopy. Both mutations affected the first base of codon 402 (glutamic acid). In patient A, a G to C transition occurred causing a glutamine substitution (GAG to CAG: E402Q) whereas in patient B, the transition was G to A yielding a lysine substitution (GAG to AAG: E402K). The sequence of the 1A helical regions of hHb1 and hHb6 as well as the 2B helical region of hHb1, were normal. Unaffected relatives did not have the hHb6 mutation and this codon was found to be highly conserved showing no alteration in the normal population (100 alleles examined). Both mutations disrupted a Taq I restriction site and restriction fragment length polymorphism analysis showed that a diagnostic 361 bp fragment could confirm the mutation. Thus, two new point mutations of the hair-specific keratin gene hHb6 have been identified in this genetic disease.

Identification of sporadic mutations in the helix initiation motif of keratin 6 in two pachyonychia congenita patients: further evidence for a mutational hot spot.

Pachyonychia congenita (PC) is a rare, autosomal dominant, ectodermal dysplasia characterized most distinctly by the presence of symmetric nail hypertrophy. In the Jadassohn-Lewandowsky form, or PC-1, additional cutaneous manifestations may include palmoplantar hyperkeratosis, hyperhidrosis, follicular keratoses, and oral leukokeratosis. Mutations have previously been identified in the 1A helix initiation motif of either keratin 6 or keratin 16 in patients with PC-1. In the current study, we have identified 2 sporadic, heterozygous mutations in the 1A helix region of the K6 isoform (K6a). The first mutation identified was a 3 base pair deletion (K6adelta N171). The second mutation was a C-to-A transversion resulting in an amino acid substitution (K6a N171K). These data, in combination with previous reports, provide further evidence that this location is a mutational hot spot.

Novel and recurrent mutations in keratin 10 causing bullous congenital ichthyosiform erythroderma.

Bullous congenital ichthyosiform erythroderma (BCIE) is a dominantly inherited keratinizing disorder characterized by erythroderma and blistering in neonates and generalized epidermolytic hyperkeratosis (EH) in adulthood. Previously, it has been shown that BCIE can be caused by mutations in either of the genes encoding K1 or K10, the keratins predominantly expressed in suprabasal layers of the epidermis. Using direct sequencing of genomic PCR fragments, we have analyzed 4 British families with BCIE, all of whom were found to carry mutations in K10. In 1 family, the affected person was found to have an unusual dinucleotide transversion mutation, 2138CC-->AA, causing two amino acid substitutions, D155E and R156S, also in the 1A domain of the K10 polypeptide. In 2 further kindreds, the previously reported "hotspot" mutations 2139C-->T and 2140G-->A were found. These mutations predict amino acid substitutions in the helix 1A domain of K10, designated R156C and R156H respectively. The proband in the fourth family was found to carry a novel mutation 4724T-->C, predicting the amino acid change L452P in the helix 2B domain of K10. All mutations were confirmed in the affected persons and were excluded from a population of 50 normal, unrelated individuals by restriction enzyme analysis. The location of these mutations in the highly conserved helix boundary motif sequences of K10 are consistent with previously reported dominant negative mutations in K10 and other keratins. Despite the unusual nature of two of these mutations, in particular the double missense mutation, the phenotypes of the affected individuals in these 4 families were entirely typical of BCIE.

Characterization and chromosomal localization of human hair-specific keratin genes and comparative expression during the hair growth cycle.

During anagen, cell proliferation in the germinative matrix of the hair follicle gives rise to the fiber and inner root sheath. The hair fiber is constructed from structural proteins belonging to four multigene families: keratin intermediate filaments, high-sulfur matrix proteins, ultra high-sulfur matrix proteins, and high glycine-tyrosine proteins. Several hair-specific keratin intermediate filament proteins have been characterized, and all have relatively cysteine-rich N- and C-terminal domains, a specialization that allows extensive disulfide cross-linking to matrix proteins. We have cloned two complete type II hair-specific keratin genes (ghHb1 and ghHb6). Both genes have nine exons and eight introns spanning about 7 kb and lying about 10 kb apart. The structure of both genes is highly conserved in the regions that encode the central rod domain but differs considerably in the C-terminal coding and noncoding sequences, although some conservation of introns does exist. These genes have been localized to the type II keratin cluster on chromosome 12q13 by fluorescence in situ hybridization. They, and their type I partner ghHa1, are expressed in differentiating hair cortical cells during anagen. In cultured follicles, ghHa1 expression declined in cortical cells and was no longer visible after 6 d, whereas the basal epidermal keratin hK14 appeared in the regressing matrix. The transition from anagen to telogen is marked by downregulation of hair cortical specific keratins and the appearance of hK14 in the epithelial sac to which the telogen hair fiber is anchored. Further studies of the regulation of these genes will improve our understanding of the cyclical molecular changes that occur as the hair follicle grows, regresses, and rests.

A novel nonepidermolytic palmoplantar keratoderma: a clinical and histopathologic study of six cases.

BACKGROUND: Some hereditary palmoplantar keratodermas (PPK) have been defined at the molecular level. OBJECTIVE: Our purpose was to establish the cause of a hereditary PPK with unique histopathologic findings in the epidermis. METHODS: Investigative studies included light and electron microscopy and determination of genomic DNA sequence. RESULTS: Six patients with PPK were found to have unique changes in the epidermis characterized by orthokeratosis, parakeratosis, perinuclear vacuolization, and keratohyalin granules that varied in size and shape and were located in the cell periphery. Electron microscopy showed the perinuclear region contained many ribosomes and vacuoles and was surrounded by a tonofibril shell. Family involvement suggested a dominant disorder. However, no mutation of keratin genes 1, 6a, 9, or 16 was found. CONCLUSION: The histopathologic features of this unique PPK most closely resemble Curth-Macklin ichthyosis for which the genetic basis has not been established. Further genetic studies are needed.

A new keratin 2e mutation in ichthyosis bullosa of Siemens.

Ichthyosis bullosa of Siemens (IBS) is a rare autosomal dominant skin condition with features similar to epidermolytic hyperkeratosis (EH). Clinical symptoms are characterized by mild hyperkeratosis with an acral distribution. Histology shows epidermolysis of upper spinous and granular cells, whereas ultrastructurally, tonofilaments form perinuclear aggregates. IBS has been linked to the type II keratin cluster on chromosome 12q, and K2e mutations have recently been identified in IBS patients. We have studied genomic DNA from two IBS families and in both cases heterozygous point mutations were found in the 2B helical domain of K2e. One family had an established mutation in codon 493 (E493K), whereas the other had an unreported mutation in the adjacent codon (E494K). Both mutations were confirmed by allele-specific PCR. These data reinforce the hypothesis that mutations in the TYRKLLEGEE motif of the 2B helix are deleterious to keratin filament network integrity and provide further evidence for the involvement of K2e mutations in IBS.

A transgenic mouse model that recapitulates the clinical features of both neonatal and adult forms of the skin disease epidermolytic hyperkeratosis.

Keratins are the major structural proteins of keratinocytes, which are the most abundant cell type in the mammalian epidermis. Mutations in epidermal keratin genes have been shown to cause severe blistering skin abnormalities. One such disease, epidermolytic hyperkeratosis (EHK), also known as bullous congenital ichthyosiform erythroderma, occurs as a result of mutations in highly conserved regions of keratins K1 and K10. Patients with EHK first exhibit erythroderma with severe blistering, which later is replaced by thick patches of scaly skin. To assess the effect of a mutated K1 gene on skin biology and to produce an animal model for EHK, we removed 60 residues from the 2B segment of HK1 and observed the effects of its expression in the epidermis of transgenic mice. Phenotypes of the resultant mice closely resembled those observed in the human disease, first with epidermal blisters, then later with hyperkeratotic lesions. In neonatal mice homozygous for the transgene, the skin was thicker, with an increased labeling index, and the spinous cells showed a collapse of the keratin filament network around the nuclei, suggesting that a critical concentration of the mutant HK1, over the endogenous MK1, was required to disrupt the structural integrity of the spinous cells. Additionally, footpad epithelium, which is devoid of hair follicles, showed blistering in the spinous layer, suggesting that hair follicles can stabilize or protect the epidermis from trauma. Blisters were not evident in adult mice, but instead they showed a thick, scaly hyperkeratotic skin with increased mitosis, resulting in an increased number of corneocytes and granular cells. Irregularly shaped keratohyalin granules were also observed. To date, this is the only transgenic model to show the typical morphology found in the adult form of EHK.

Keratins (K16 and K17) as markers of keratinocyte hyperproliferation in psoriasis in vivo and in vitro.

Keratinocyte differentiation in psoriasis was examined using a panel of monospecific monoclonal antibodies to keratins (K), including two recently developed monoclonal antibodies raised to carboxy terminal peptides of K6 (LL020) and K16 (LL025). Keratinocytes from normal skin, untreated psoriatic plaques and non-lesional psoriatic skin, were cultured using multiple in vitro systems. Time-lapse cinephotography was used to measure the intermitotic time of normal and psoriatic keratinocytes in both low calcium-defined and serum-containing media. The intermitotic time did not differ significantly between psoriatic and normal keratinocytes. Keratin expression of psoriatic and normal keratinocytes in vitro was examined by both gel electrophoresis and immunocytochemistry. K6, K16 and K17 were detected suprabasally in all culture systems in vitro, but only in interfollicular psoriatic epidermis in vivo, and not in normal skin. Small subpopulations of keratinocytes expressed simple epithelial keratins K7, K8, K18 and K19 in cultures on plastic substrates, but these keratins were absent in skin equivalents of normal or psoriatic skin. No psoriasis-specific pattern of differentiation was found in vitro. As the K6 peptide antibody reacted with basal cells of normal skin, probably due to K5 cross-reactivity, K16 expression determined by LL025 was found to be the most sensitive indicator of the psoriatic state of differentiation, and this antibody is recommended for future work on psoriasis. K17 had a distinct pattern of tissue distribution in normal skin: K17, but not K16, was present in basal myoepithelial cells in sweat glands, and the deep outer root sheath, but K17 distribution paralleled that of K16 in suprabasal psoriatic epidermis. As keratins K6, K16 and K17 are expressed in keratinocyte hyperproliferation, when high levels of certain cytokines are also expressed, the role of growth factors and regulatory nuclear transcription factors in the control of K6, K16 and K17 expression in psoriasis requires further study, in order to provide insight into the relationship between proliferation and differentiation.

Mutation of a type II keratin gene (K6a) in pachyonychia congenita.

Pachyonychia congenita (PC) is a rare autosomal dominant condition characterized by multiple ectodermal abnormalities. Patients with Jadassohn-Lewandowsky Syndrome (MIM #167200; PC-1) have nail defects (onchyogryposis), palmoplantar hyperkeratosis, follicular hyperkeratosis and oral leukokeratosis. Those with the rarer Jackson-Lawler Syndrome (MIM #167210; PC-2) lack oral involvement but have natal teeth and cutaneous cysts. Ultra-structural studies have identified abnormal keratin tonofilaments and linkage to the keratin gene cluster on chromosome 17 has been found in PC families. Keratins are the major structural proteins of the epidermis and associated appendages and the nail, hair follicle, palm, sole and tongue are the main sites of constitutive K6, K16 and K17 expression. Furthermore, mutations in K16 and K17 have recently been identified in some PC patients. Although we did not detect K16 or K17 mutations in PC families from Slovenia, we have found a heterozygous deletion in a K6 isoform (K6a) in the affected members of one family. This 3 bp deletion (AAC) in exon 1 of K6a removes a highly conserved asparagine residue (delta N170) from position 8 of the 1A helical domain (delta N8). This is the first K6a mutation to be described and this heterozygous K6a deletion is sufficient to explain the pathology observed in this PC-1 family.

Sequence and expression of human hair keratin genes.

Normal hair growth and differentiation requires co-ordinate expression of many hair specific structural protein genes. It has been established that one of the 4 major groups of hair structural proteins, low-sulphur hair keratins, belongs to the intermediate filament (IF) multigene family. Hair keratin IF proteins differ from those of other epithelia as they contain cysteine-rich terminal domains allowing more extensive disulphide bonding to the high-sulphur hair matrix proteins. Until recently, little information concerning the primary sequence of hair keratins was available but cloning of some mouse hair and sheep wool keratins has now been reported. Using these sequences, we have polymerase chain reaction (PCR) amplified genomic fragments of human hair-specific keratin IF genes and isolated cosmid clones containing full length genes. We have sequenced part of these genes and studied their expression in human hair follicles. Hair specific keratin fragments were amplified from placental gDNA by PCR primed with synthetic oligonucleotides. Fragments were cloned and sequenced after ligation into pGEM-3Z and labelled riboprobes were generated for in situ hybridization on human skin sections. A human cosmid library was screened with PCR fragments and clones encoding human hair keratin genes were characterised by southern hybridization and sequencing. The type I human hair-specific keratin clones obtained (HaKA1-b2, 386 bp; hHaKA1-XH1, 1202 bp) encoded 2B helix, C-terminal and 3'nc regions and were 65% homologous to mouse sequences. The type II hair keratin clone (hHaKB2-1, 829 bp) also encoded 2B helix and C-terminal regions and was 95% homologous to mouse. In situ hybridization on human skin sections showed a specific reaction with precortical cells of the hair follicle. One human cosmid clone, isolated with the hHaKB2-1 probe, contained two type II hair keratin genes about 7 kb apart, each of which had 9 exons spanning approximately 6 kb. The coding sequences were homologous to mouse cDNA (77-88%). These human hair-specific keratin clones are useful molecular tools for studies of hair differentiation.

Down-regulation of keratin 14 gene expression after v-Ha-ras transfection of human papillomavirus-immortalized human cervical epithelial cells.

Keratin expression in human cervical squamous cell carcinoma (SCC) lines differed significantly from both normal and human papillomavirus (HPV) immortalized exocervical cells. Keratin 14 (K14) expression, determined by protein synthesis and mRNA levels, was dramatically down-regulated in the cervical SCC lines while keratin 5 (K5) expression was not. K14 expression was similarly down-regulated in an HPV-16 immortalized cervical cell line after tumorigenic transformation with recombinant v-Ha-ras DNA. Cultures derived from nude mouse tumor explants also exhibited an altered keratin profile and the levels of K14 protein synthesis, as well as K14 mRNA, were not detectable. In both cases K5 protein synthesis was not significantly down-regulated. In addition, neoplastic cervical SCC lines exhibited up-regulation of keratins 7, 8, 13, and 19, combined with slight down-regulation of keratins 6 and 16. Epidermal keratinocytes responded in a different manner to exocervical cells. Transfection of human papillomavirus-immortalized epidermal keratinocytes with the BglII N fragment of herpes simplex virus 2 produced a neoplastic cell line, but K5 and K14 expression remained unchanged. Thus, neoplastic transformation of human exocervical cells, both in vivo (spontaneous cervical SCC) and in vitro (HPV-16- and v-Ha-ras-induced cervical SCC), is accompanied by characteristic changes in keratin expression. The specific down-regulation of K14 in these tumorigenic cervical cells, in the absence of significant changes in the expression of K5, implies that the normal coordinate regulation of K5 and K14 gene expression has been uncoupled.

Human bronchial epithelial cells transformed by the c-raf-1 and c-myc protooncogenes induce multidifferentiated carcinomas in nude mice: a model for lung carcinogenesis.

We have previously described the neoplastic transformation of immortalized human bronchial epithelial cells (BEAS-2B) by the combination of the c-raf-1 and c-myc protooncogenes and the concomitant induction of neuron-specific enolase mRNA expression (A. Pfeifer et al., Proc. Natl. Acad. Sci. USA, 86: 10075-10079, 1989). In this paper we describe the morphological, biochemical, and immunohistochemical characteristics of the primary c-raf-1/c-myc tumors, xenografts of these tumors, and tumors that originated from cell lines of the primary neoplasm. The tumors were morphologically characterized by the appearance of desmosomes and tonofilaments, microvilli, and dense core granules representing markers of squamous, glandular, and neuroendocrine differentiation, respectively. A total of 11 of 13 tumors were positive by immunohistochemical techniques for neuron-specific enolase, serotonin (nine of 13), and calcitonin (six of 13). Keratins were expressed in 11 of 13 tumors, and while specific keratins (K5, K7, K16/K17) decreased, there was an increase of vimentin in the tumor cells. Gastrin-releasing peptide immunoreactivity was detectable in a small number of tumors (five of 13). BEAS-2B cells transfected with the c-raf-1 and c-myc protooncogenes and cell lines established from the primary tumors expressed major histocompatibility Class II antigen which has been found on small cell lung carcinoma cells. The tumors induced by the c-raf-1 and c-myc protooncogenes resemble the multidifferentiated phenotype of small cell lung cancer frequently detected in vivo and present a defined model to study the relation between molecular markers, phenotypical appearance, and response to chemotherapeutic agents and radiation.