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Identification of differentially expressed genes in chemically induced skin tumors
Abstract
Previous studies have demonstrated a role for the fos gene in promoting malignant conversion of mouse skin tumors. In the study reported here, differential display was performed to identify fos- and jun-regulated genes that are differentially expressed during premalignant progression. Total RNA isolated from variants of the papilloma cell line SP-1 transduced with retroviral vectors expressing v-jun and v-fos alone or in tandem was analyzed for the presence of differentially expressed transcripts by using 35 different primer combinations. Differentially expressed clones were rescreened by dot-blot analysis by using cDNA from chemically induced tumors with a high or low risk of malignant conversion. Three differentially displayed fragments were isolated in this analysis. Homology searches indicated that these fragments shared significant homology with the apoptosis inhibitor bcl-2, human alternative splicing factor/splicing factor 2 (ASF/SF2), and a novel gene not present in the GenBank or EMBL databases. In situ hybridization indicated that the expression levels of the bcl-2 homolog increased with malignant potential in chemically derived mouse skin tumors. A similar analysis indicated that expression of the ASF/SF2 homolog was greater in papillomas than in normal skin or in squamous cell carcinomas. Transcripts for this gene were most abundant in the granular layer. The expression pattern of the third differential display fragment was consistent with that of a tumor suppressor gene. This gene was expressed at very high levels in normal skin and benign papillomas but was essentially undetectable in squamous cell carcinomas. Through this approach, we identified known and novel genes that may contribute to malignant progression in epidermal tumors.
... In transgenic mice, a skin-targeted Jun mutant blocks skin tumor formation by the two-stage chemical carcinogenesis protocol (12) or by UVB radiation (13). Moreover, AP1 DNA-binding activity is increased in nuclear extracts from chemically induced papillomas that have a high risk for malignant progression (14). Transduction of v-ras Ha keratinocytes with v-Fos results in malignant conversion (15), and malignant skin tumors do not develop in response to Ras gene activation and tumor promoter treatment in c-Fos null mice (16). ...
... The exposure of keratinocytes to UVB radiation transiently up-regulates FOS and JUN family proteins, whereas their transcripts increase at later times. AP1 factors are upregulated and/or activated during skin carcinogenesis (14), in response to treatment with chemical promoters such as TPA (40) or physical tumor-promoting agents such as UVB radiation (5,41), or upon transduction of oncogenes such as v-ras Ha in primary keratinocytes (15,42). AP1 factor activation is necessary for the transformation in epidermal cells (42). ...
... AP1 factor activation is necessary for the transformation in epidermal cells (42). Interestingly, AP1 DNA-binding activity is higher in nuclear extracts from papillomas at high risk of malignant progression (14), and conversely, retinoid receptor protein levels are more reduced in skin tumors at higher risk for malignant progression (30). Apparently, retinoid receptor and AP1 signaling pathways are interrelated. ...
The main cause of skin cancer and photo-aging is chronic exposure to ultraviolet B (UVB) radiation. Such damage can be ameliorated by retinoid treatment. UVB-radiation-induced skin carcinogenesis is associated with the induction of activator protein 1 (AP1) signaling and factors, namely FOS and JUN family members. We investigated the effects of several retinoids, all-trans-retinoic acid (tRA), 9-cis-retinoic acid (cRA), and N-(4-hydroxyphenyl)-retinamide (HPR), on UVB-induced damage in primary mouse keratinocytes. In addition, the interplay between UVB radiation, retinoid receptors, and AP1 signaling was assessed using Western blot analysis and ribonuclease protection and gene reporter assays. Exposure of keratinocytes to UVB radiation caused a down-regulation of the retinoid receptor protein levels in a proteasome-mediated manner. In contrast, FOS and JUN proteins were transiently induced shortly after exposure to UVB radiation. Retinoid treatment caused a dose-dependent reduction in the levels of retinoid receptor proteins. When irradiated cells were treated with retinoids, no significant effects on AP1 protein expression were noted. Interestingly, pretreatments with tRA and cRA, but not HPR, suppressed UVB-radiation-induced AP1 activity by more than 50%, whereas post-treatment failed to produce similar effects. Our findings indicate that the inhibition of AP1 activity by retinoids explains, at least in part, the chemopreventive potential of retinoids in UV-radiation-associated epidermal damage.
... AP-1 activity contributes to malignant progression of epidermal cells, and AP-1 DNA binding activity is increased in nuclear extracts from chemically induced papillomas that have a high risk of malignant progression (10). Dong et al. (11) showed that AP-1 transactivation is required for tumor promoter-induced transformation, as measured by anchorage-independent growth in JB6 cells. ...
... The late markers, loricrin and filaggrin, are induced more rapidly and intensely in v-ras Ha keratinocytes (18), reminiscent of the expression of these markers in keratinocytes treated with the phorbol ester, 12-O-tetradecanoyl-phorbol acetate, an activator of PKC (19). Moreover, there is an increase in PKC␣ activity in v-ras Ha keratinocytes (18), and the changes in marker gene expression seen in these cells can be reversed through the use of pharmacological inhibitors of PKC (18) or PKC␣ antisense oligonucleotides (10). We have shown that certain members of the AP-1 family are regulated in a PKC-dependent manner in differentiating keratinocytes (21). ...
The induction of mouse skin papillomas by initiation-promotion protocols is associated with aberrant expression of epithelial markers in the tumor mass. Similarly, initiation of mouse keratinocytes with a retrovirus encoding the v-rasHa gene (v-rasHa keratinocytes) causes characteristic alterations of epidermal gene expression (A. A. Dlugosz et at, Cancer Res., 54: 6413-6420, 1994). Because activator protein 1 (AP-1) proteins are likely targets of Ras activation, we have examined the role of AP-1 factors in v-rasHa keratinocytes. Introduction of v-rasHa into keratinocytes up-regulates c-Fos, deltaFos B, and Fra-1 transcripts and protein levels in nuclear extracts. The expression of Jun proteins is not significantly altered in v-rasHa keratinocytes. Transduction of cells with v-rasHa results in increased AP-1-dependent transcriptional activity, which is also simulated by transfection of keratinocytes with either c-Fos or deltaFos B but not Fra-1, suggesting that the up-regulation of c-Fos and deltaFos B contributes to this effect. To explore the role of AP-1 proteins in regulating keratinocyte markers in v-rasHa keratinocytes, we blocked the binding of AP-1 proteins to DNA by infecting keratinocytes with an adenovirus encoding a dominant-negative Fos mutant (A-FOS). A-FOS replaces endogenous Fos proteins in the formation of heterodimers with Jun family members and thus prevents the AP-1 transcription factor from binding to DNA. In v-rasHa keratinocytes, the A-FOS virus reversed the suppression of keratins 1 and 10 transcripts and protein, which is characteristically seen in tumors and v-rasHa keratinocytes. A-FOS also increased protein levels but reduced transcripts for the late marker, loricrin, a component of the cornified envelope. These findings indicate that AP-1 proteins are involved in the changes in gene expression that define the v-rasHa phenotype in mouse keratinocytes.
... Furthermore, overexpression of a mouse homolog to the anti-apoptotic protein Bcl-2 was detected in the basal layer of papillomas and in most of the cells of the carcinomas (Rutberg, Lee et al. 1997). ...
... Blocking of AP-1 activity in Ras-transformed cells has been shown to suppress transformation (Lloyd et al., 1991). Enhanced AP-1 activity is frequently associated with in vivo and in vitro models of skin cancer (Rutberg et al., 1997;Domann et al., 1994). ...
The MGSA/GRO protein is endogenously expressed in almost 70% of the melanoma cell lines and tumors, but not in normal melanocytes. We have previously demonstrated that over-expression of human MGSA/GROalpha, beta or gamma in immortalized murine melanocytes (melan-a cells) enables these cells to form tumors in SCID and nude mice. To examine the possibility that the MGSA/GRO effect on melanocyte transformation requires expression of other genes, differential display was performed. One of the mRNA's identified in the screen as overexpressed in MGSA/GRO transformed melan-a clones was the newly described M-Ras or R-Ras3 gene, a member of the Ras gene superfamily. Over-expression of MGSA/GRO upregulates M-Ras expression at both the mRNA and protein levels, and this induction requires an intact glutamine-leucine-arginine (ELR)-motif in the MGSA/GRO protein. Western blot examination of Ras expression revealed that K- and N-Ras proteins are also elevated in MGSA/GRO-expressing melan-a clones, leading to an overall increase in the amount of activated Ras. MGSA/GRO-expressing melan-a clones exhibited enhanced AP-1 activity. The effects of MGSA/GRO on AP-1 activation could be mimicked by over-expression of wild-type M-Ras or a constitutively activated M-Ras mutant in control melan-a cells as monitored by an AP-1-luciferase reporter, while expression of a dominant negative M-Ras blocked AP-1-luciferase activity in MGSA/GRO-transformed melan-a clones. In the in vitro transformation assay, over-expression of M-Ras mimicked the effects of MGSA/GRO by inducing cellular transformation in control melan-a cells, while over-expression of dominant negative M-Ras in MGSA/GROalpha-expressing melan-a-6 cells blocked transformation. These data suggest that MGSA/GRO-mediated transformation requires Ras activation in melanocytes.
... Recently, a partial catalog of secreted proteins in keratinocyte cultures was published by Katz and Taichman (1999). No systematic studies on keratinocyte gene expression at the mRNA level have been undertaken so far, although a considerable amount of data is available from randomly sequenced keratinocyte cDNA libraries (Konishi et al, 1994), differential display polymerase chain reaction (PCR) (Frank and Werner, 1996;Frank et al, 1997;Munz et al, 1997;Rivas et al, 1997;Rutberg et al, 1997;DiSepio et al, 1998) and cDNA ®lter arrays (Trenkle et al, 1998). ...
Keratinocyte gene expression was surveyed more comprehensively than before, by means of serial analysis of gene expression. A total of 25,694 tags derived from expressed mRNA, were analyzed in a model for normal differentiation and in a model where cultured keratinocytes were stimulated for a prolonged period of time with tumor necrosis factor-alpha, thus mimicking aberrant differentiation in the context of cutaneous inflammation. Serial analysis of gene expression revealed many transcripts derived from unknown genes and a large number of genes that are not known to be expressed in keratinocytes; furthermore, these data provide quantitative information about the relative abundance of transcripts, allowing the identification of differentially expressed genes. A major part of the identified transcripts accounted for genes involved in energy metabolism and protein synthesis. A large proportion of all transcripts (6%) corresponded to genes associated with terminal differentiation and barrier formation. Another highly expressed functional group of genes (2% of all transcripts) corresponded to proteins involved in host protection such as antimicrobial proteins and proteinase inhibitors. Three of these genes were not known to be expressed in keratinocytes, and some were upregulated after prolonged tumor necrosis factor-alpha exposure. Our data on expressed genes in keratinocytes are consistent with the known function of human epidermis, and provide a first step to generate a transcriptome of human keratinocytes.
... 28 Upregulation of SF2 in chemically-induced skin tumors has been previously shown. 29 In our system, SF2 was upregulated in all immortal cells, including choriocarcinomas, suggesting that post-transcriptional regulation of certain mRNAs may play a role in tumorigenesis. ...
Tumorigenesis results from genetic alterations that occur in a stepwise manner giving rise to cells with increasingly cancer-like characteristics. We used in vitro propagated first trimester human extravillous trophoblast (EVT) cells to identify genetic changes responsible for the transition of the EVT from a normal to premalignant stage. The model used consisted of a normal invasive EVT (HTR8) cell line and its premalignant derivative (RSVT2/C) generated by transfection with the SV40 Tag and selected using a forced crisis regimen. RSVT2/C display increased proliferative, migratory and invasive behavior, unresponsiveness to anti-proliferative and anti-invasive signals of TGFbeta and a deficiency in gap junctional intercellular communication. These cells, however, were unable to form colonies on soft agar or tumors in nude mice and are thus defined as premalignant. Differential display revealed 18 gene sequences, 7 with unknown and 11 with known identity, showing altered expression between the normal HTR8 and premalignant RSVT2/C cell lines. The known sequences include the potential tumor suppressors insulin-like growth factor binding protein (IGFBP)-5 and fibronectin (FN) and potential protooncogenes such as chromokinesin (KIF4), alternative splicing factor (SF2), dynein, DNA polymerase epsilon (DNApol epsilon) and NF-kappaB activating kinase (NAK). The role of the remaining 4 genes upregulated in the premalignant EVT is presently unknown and these are FK506 binding protein (FKBP) 25, histone protein (HP1Hs)-gamma, nucleoporin (Nup) 155 and an 82 kDa acidic human protein. The functional role of IGFBP-5 was examined in the control of proliferation, migration and invasiveness of RSVT2/C cells measured in vitro. IGFBP-5 alone had no effect on these properties of RSVT2/C cells. Furthermore, unlike normal EVT cells, RSVT2/C cells exhibited refractoriness to the migration stimulating signals of IGF-II, which was explained by the loss or downregulation of the IGF type 2 receptor (IGF-R2). RSVT2/C cells, however, expressed the IGF type 1 receptor (IGF-R1) and responded to IGF-I by increased proliferation. This response was blocked with increasing concentrations of IGFBP-5. These results suggest that the loss of IGFBP-5 and possibly IGF-R2, both of which can sequester IGF-I from IGF-R1, permits unhindered proliferation of the premalignant EVT in an IGF-I rich environment of the fetal-maternal interface. The functions of the other differentially expressed genes, some of which are essential for cell cycle progression or cell survival require further investigation.
... Five genes with potential oncogenic function were upregulated in all immortal cell lines including premalignant and malignant trophoblast cells. These are chromokinesin (KIF 4), which is involved in cell cycle progression (Wang and Adler 1995); alternative splicing factor (SF)2, involved in alternative splicing of certain tumor suppressor genes (Rutberg et al. 1997); dynein, involved in mitotic spindle formation (Ma et al. 1999); human DNA polymerase ⑀ p12 subunit, involved in DNA replication and repair ; and NFκB activity kinase (NAK), involved in the promotion of cell survival (Burrow et al. 2000). Functions of four genes, FK 506 binding protein (FKBP)25, histone protein (HP1Hs)-γ, nucleoporin (Nup)155, and a 82 KDa acidic human protein, which were upregulated in immortal cell lines, remain completely unknown at present. ...
The human placenta is a highly invasive tumor-like structure in which a subpopulation of placental trophoblast cells known as the "extravillous trophoblast" (EVT) invades the uterine decidua and its vasculature to establish adequate fetal-maternal exchange of molecules. By utilizing in vitro-propagated short-lived EVT cell lines we found that molecular mechanisms responsible for their invasiveness are identical to those of cancer cells; however, unlike cancer cells, their proliferation, migration, and invasiveness in situ are stringently controlled by decidua-derived transforming growth factor (TGF)-beta. By SV40T antigen transfection of normal EVT cells followed by a forced crisis regimen in culture we produced an immortalized premalignant derivative that is hyperproliferative, hyperinvasive, and deficient in gap-junctional intercellular communication. Both premalignant and malignant EVT (JAR and JEG-3 choriocarcinoma) cell lines were found to be TGF-beta-resistant. Using these cell lines, we investigated genetic changes responsible for transition of the normal EVT cells to premalignant and malignant phenotype. Hyperinvasiveness in both cases resulted from a downregulation of tissue inhibitor of metalloprotease (TIMP)-1 and plasminogen activator inhibitor (PAI)-1 genes. In contrast to normal EVT cells, both cell types failed to upregulate these genes in response to TGF-beta. Loss of TGF-beta response in malignant EVT cells was explained by the loss of expression of Smad3 gene. Differential mRNA display of normal and premalignant EVT cells identified up- and down-regulation of numerous known or novel genes in premalignant EVT cells, with potential oncogenic and (or) tumor-suppressor functions, e.g., loss of fibronectin and insulin-like growth factor binding protein (IGFBP-5). Premalignant EVT cells also lost IGF receptor type 2 (IGFR-II). IGFBP-5 was shown to be a negative regulator of IGF-1-induced proliferation of premalignant EVT cells, so that loss of IGFBP-5 as well as IGFR-II permitted their unrestricted proliferation in an IGF-I-rich microenvironment of the fetal-maternal interface. The present model may be a good prototype for identifying genetic changes underlying epithelial tumor progression.
... In addition, Katz et al. recently published a partial catalog of secreted proteins in keratinocyte cultures [14]. Thus far, no systematic studies on keratinocyte gene expression at the mRNA level have been undertaken, although considerable data are available from randomly sequenced keratinocyte cDNA libraries [15,16], differentialdisplay PCR [17][18][19][20][21][22], and cDNA filter arrays [23]. ...
Serial analysis of gene expression (SAGE) is a powerful technique for global expression profiling without prior knowledge of the genes of interest. We carried out SAGE analysis of purified keratinocytes derived from human skin biopsy specimens, resulting in a partial transcriptome of human epidermis. We identified 7645 unique SAGE tags with quantitative information from 15,131 collected SAGE tags obtained from approximately 3 x 10(6) epidermal cells. This catalog contains a large number of genes that were not previously known to be expressed by human epidermis. Comparison with the databases of all known human SAGE tags allowed us to identify a number of keratinocyte-specific tags that putatively correspond to formerly unknown genes. Surprisingly, human epidermal keratinocytes in vivo show relatively low expression levels of genes typically associated with epidermal differentiation, whereas the expression levels of housekeeping genes are considerably higher than in cultured keratinocytes. This study provides a first step toward a transcriptome of human epidermis and, as such, harbors a wealth of information to identify genes involved in skin function, and candidate genes for genetic skin disorders.
Differential-display PCR (DD-PCR), a comparative method for detecting alterations in the expression pattern of unknown genes, was applied to identify markers for environmental pollution of the coastal marine environment, in Donax trunculus (Mollusca: Bivalvia). Bivalves collected from a site typified by industrial pollution were compared with bivalves collected from an unpolluted site. Additionally, bivalves from the unpolluted site were exposed in the laboratory to increasing levels of pollution. Multiple responsive fragments, including qualitative and quantitative differentials, were observed in both experimental designs. Fragment-specific primers may be designed for use in diagnostic amplifications, according to the sequence of selected differential fragments. This study is a first step toward the establishment of a marine warning and monitoring system based on the most basic level of biological response.
Tumorigenesis results from genetic alterations that occur in a stepwise manner giving rise to cells with increasingly cancer-like characteristics. We used in vitro propagated first trimester human extravillous trophoblast (EVT) cells to identify genetic changes responsible for the transition of the EVT from a normal to premalignant stage. The model used consisted of a normal invasive EVT (HTR8) cell line and its premalignant derivative (RSVT2/C) generated by transfection with the SV40 Tag and selected using a forced crisis regimen. RSVT2/C display increased proliferative, migratory and invasive behavior, unresponsiveness to anti-proliferative and anti-invasive signals of TGFβ and a deficiency in gap junctional intercellular communication. These cells, however, were unable to form colonies on soft agar or tumors in nude mice and are thus defined as premalignant. Differential display revealed 18 gene sequences, 7 with unknown and 11 with known identity, showing altered expression between the normal HTR8 and premalignant RSVT2/C cell lines. The known sequences include the potential tumor suppressors insulin-like growth factor binding protein (IGFBP)-5 and fibronectin (FN) and potential protooncogenes such as chromokinesin (KIF4), alternative splicing factor (SF2), dynein, DNA polymerase ϵ (DNApol ϵ) and NF-κB activating kinase (NAK). The role of the remaining 4 genes upregulated in the premalignant EVT is presently unknown and these are FK506 binding protein (FKBP) 25, histone protein (HP1Hs)-γ, nucleoporin (Nup) 155 and an 82 kDa acidic human protein. The functional role of IGFBP-5 was examined in the control of proliferation, migration and invasiveness of RSVT2/C cells measured in vitro. IGFBP-5 alone had no effect on these properties of RSVT2/C cells. Furthermore, unlike normal EVT cells, RSVT2/C cells exhibited refractoriness to the migration stimulating signals of IGF-II, which was explained by the loss or downregulation of the IGF type 2 receptor (IGF-R2). RSVT2/C cells, however, expressed the IGF type 1 receptor (IGF-R1) and responded to IGF-I by increased proliferation. This response was blocked with increasing concentrations of IGFBP-5. These results suggest that the loss of IGFBP-5 and possibly IGF-R2, both of which can sequester IGF-I from IGF-R1, permits unhindered proliferation of the premalignant EVT in an IGF-I rich environment of the fetal-maternal interface. The functions of the other differentially expressed genes, some of which are essential for cell cycle progression or cell survival require further investigation.
PRKAR1A inactivation leads to dysregulated cAMP signaling and Carney complex (CNC) in humans, a syndrome associated with skin, endocrine and other tumors. The CNC phenotype is not easily explained by the ubiquitous cAMP signaling defect; furthermore, Prkar1a+/- mice did not develop skin and other CNC tumors. To identify whether a Prkar1a defect is truly a generic but weak tumorigenic signal that depends on tissue-specific or other factors, we investigated Prkar1a+/- mice when bred within the Rb1+/- or Trp53+/- back-grounds, or treated with a two-step skin carcinogenesis protocol. Prkar1a+/- Trp53+/- mice developed more sarcomas than Trp53+/- mice (P < 0.05) and Prkar1a+/- Rb1+/- mice grew more (and larger) pituitary and thyroid tumors than Rb1+/- mice. All mice with double heterozygosity had significantly reduced life-spans compared with their single-heterozygous counterparts. Prkar1a+/- mice also developed more papillomas than wild-type animals. A whole-genome transcriptome profiling of tumors produced by all three models identified Wnt signaling as the main pathway activated by abnormal cAMP signaling, along with cell cycle abnormalities; all changes were confirmed by qRT-PCR array and immunohistochemistry. siRNA down-regulation of Ctnnb1, E2f1 or Cdk4 inhibited proliferation of human adrenal cells bearing a PRKAR1A-inactivating mutation and Prkar1a+/- mouse embryonic fibroblasts and arrested both cell lines at the G0/G1 phase of the cell cycle. In conclusion, Prkar1a haploinsufficiency is a relatively weak tumorigenic signal that can act synergistically with other tumor suppressor gene defects or chemicals to induce tumors, mostly through Wnt-signaling acti-vation and cell cycle dysregulation, consistent with studies in human neoplasms carrying PRKAR1A defects. © The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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This study used the induction of squamous cell carcinomas on mouse skin as an experimental model to evaluate molecular and biochemical changes that contribute to the neoplastic phenotype. The study was facilitated by the development of keratinocyte cell culture assays that reproduce each stage of the carcinogenesis process, by discoveries of stage-specific genetic and epigenetic changes and by application of pharmacological and molecular tools that modify each step. An early event in the transformation of keratinocytes involves mutation and activation of the rasHa gene, producing a benign tumor. The phenotypic consequences of ras mutations are mediated by activation of the epidermal growth factor receptor (EGFR), upregulation of protein kinase C (PKC) alpha and AP-1 mediated transcriptional activity and inactivation of PKC delta through tyrosine phosphorylation. These changes in benign tumors are manifested by hyperproliferation (EGFR), aberrant expression of keratinocyte genes (PKC alpha and AP-1) and delayed terminal differentiation (PKC delta). Accumulated chromosomal abnormalities, multifocal phenotypic changes and alterations in gene expression are associated with premalignant progression. Upregulation of the fos gene and AP-1 transcriptional activity causes malignant conversion of benign keratinocytes. In the absence of c-fos, benign tumor cells fail to upregulate secreted angiogenic and proteolytic factors and this may prevent malignant conversion. These pathways provide targets for preventive strategies to interrupt the process of carcinogenesis prior to the evolution of the fully malignant tumor.
To identify differentially expressed genes in a human lens epithelial cell line exposed to oxidative stress.
Reverse transcriptase-polymerase chain reaction (RT-PCR) differential display was used to evaluate differential gene expression in a human lens epithelial cell line (SRA 01-04) when cells were exposed for 3 hours to a single bolus of 200 microM hydrogen peroxide. Differentially expressed genes were identified through DNA sequencing and a nucleotide database search. Differential expression was confirmed by northern blot and RT-PCR analyses.
Using 18 primer sets, 28 RT-PCR products were differentially expressed between control and hydrogen peroxide-treated cells. In stressed cells, mitochondrial transcripts nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4 and cytochrome b were downregulated 4-fold. Of the cytoplasmic mRNAs, glutamine cyclotransferase decreased 10-fold, whereas cytokine-inducible nuclear protein, alternative splicing factor 2, and beta-hydroxyisobutyryl-coenzyme A hydrolase increased 2-, 4-, and 10-fold, respectively. Analysis of mitochondrial transcripts in a 24-hour time course showed that NADH dehydrogenase subunit 4 mRNA decreased by 2-fold as early as 1 hour after oxidative stress, whereas the rate of decrease was slower for cytochrome b, cytochrome oxidase III, and 16S rRNA.
Oxidative stress induced specific expressed gene changes in hydrogen peroxide-treated lens cells, including genes involved in cellular respiration and mRNA and peptide processing. These early changes may reflect pathways involved in the defense, pathology, or both of the lens epithelium, which is exposed to oxidative stress throughout life.
The Armed Forces Institute of Pathology (AFIP) is well known for providing expert pathology in many specialties and educational courses for civilian and military personnel. Some of the departments at the AFIP have also developed expertise in various advanced laboratory techniques for diagnosis and research that are applicable to dermatology and are not available at most medical centers.
The differential display of cDNA species defined by a combination of so-called anchored and arbitrary primers has been acknowledged as a powerful complex strategy to identify differences in gene expression, and depends in its original version, inaugurated by P. Liang and A. B. Pardee in 1992, on the use of radioactive-labelled nucleotides. As a non-radioactive methodological alternative, we established the use of polyesterfilm-backed 10% polyacrylamide gels for horizontal differential-display electrophoresis under non-denaturing conditions, with subsequent detection of cDNA bands by an optimized, semi-automated silver staining omitting any fixation step. Polyacrylamide gel slices carrying the silvered cDNA species of interest were cut out, chopped, squashed and incubated in an ammonium acetate/EDTA solution at 37 degrees C overnight under vigorous shaking. This procedure resulted in a 70% average success rate for subsequent PCR reamplification with regard to the number of cDNAs harvested from the differential-display gel. Novel sequence data of three cDNA clones are communicated, which under these methodological conditions were selected to be up- or down-regulated, respectively, by antipsoriatic dithranol in cultured HaCaT keratinocytes.
Concern exists regarding peroxisome proliferator (PP) xenobiotic exposure because many PPs are potent hepatocarcinogens in rodents. The mechanism of carcinogenicity induced by PPs is atypical compared with those of other hepatocarcinogens in that the former appears to involve alterations in expression of PP-activated receptor (PPAR) target genes rather than direct mutagenicity. To begin to identify some of these genes, we used differential display to compare mRNA expression between hepatic adenomas and adjacent non-tumor liver from rats fed the potent PP Wy-14643 (WY) for 78 wk. Here, we report increased expression of the acute-phase protein (APP) gene alpha-1 antitrypsin (AT) and decreased expression of alpha2-urinary globulin in the tumors. Similar changes were seen in hepatic adenomas induced by a diethylnitrosamine and phenobarbital protocol, indicating a lack of specificity for PP-induced tumors. Additional APP genes, including ceruloplasmin, haptoglobin, beta-fibrinogen, and alpha1-acid glycoprotein were also upregulated in WY-induced tumors but were downregulated in the livers of rats administered a different PP for 13 wk. Mice treated with either WY or di(2-ethylhexyl) phthalate for 3 wk had decreased hepatic AT expression but increased expression of ceruloplasmin and haptoglobin. PPARalpha-null mice showed no hepatic APP gene alteration after PP treatment but had higher basal expression than did wild-type controls. We conclude that PPARalpha activation by several different PPs leads to dysregulation of hepatic APP gene expression in rats and mice. This dysregulation may indicate alterations in cytokine signaling networks regulating both APP gene expression and hepatocellular proliferation.
Alterations leading to inactivation of tumor suppressor genes are often associated with reduced levels of tumor suppressor gene expression. Specific tumors or cell lines may exhibit deletion of one or both gene copies, promoter methylation, splice-site mutations, nonsense mutations that induce premature translational termination and destabilize mRNA transcripts, or a combination of these. Such mutations result in complete absence or partial reduction in levels of tumor suppressor mRNA. Therefore, cDNA subtraction has been frequently used for identification of candidate tumor suppressor genes (1–7). As multiple tumor suppressor genes function in signaling pathways that regulate expression of oncogene transcription factors (8), subtraction methods are also useful for identification of downstream targets of these pathways. For example, embryo cells can be derived from mice engineered to be deficient in both alleles of a tumor suppressor. Then primary cell cultures can be analyzed by comparison with littermate control cells for identification of candidate target genes.
The epidermis protects the organism against physical, chemical and biological challenges, and it acts as a signalling interface
between the environment and the body. In order to perform these functions, the epidermal keratinocytes express a wide range
of genes, several of which have been characterised previously. Recently, significant progress has been made in the large-scale
analysis of keratinocyte gene expression, enabling a more profound insight into keratinocyte biology and human skin diseases.
Transcriptome analysis — serial analysis of gene expression (SAGE) and microarrays — and proteome analysis have been performed
on intact human epidermis and on keratinocytes cultured in model systems that mimic normal and diseased human epidermis. Here,
we review the current state of large-scale gene expression analysis of human skin, with an emphasis on SAGE and complementary
DNA microarrays. The merits and limitations of various approaches (transcriptomics versus proteomics) are discussed and the
practical issues such as sample preparation from skin biopsies, and the use of in vitro models are briefly addressed.
SF2 is a protein factor essential for constitutive pre-mRNA splicing in HeLa cell extracts and also activates proximal alternative 5' splice sites in a concentration-dependent manner. This latter property suggests a role for SF2 in preventing exon skipping, ensuring the accuracy of splicing, and regulating alternative splicing. Human SF2 cDNAs have been isolated and overexpressed in bacteria. Recombinant SF2 is active in splicing and stimulates proximal 5' splice sites. SF2 has a C-terminal region rich in arginine-serine dipeptides, similar to the RS domains of the U1 snRNP 70K polypeptide and the Drosophila alternative splicing regulators transformer, transformer-2, and suppressor-of-white-apricot. Like transformer-2 and 70K, SF2 contains an RNP-type RNA recognition motif.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
The expressed gene coding for mouse ribosomal protein L7 (rpL7) was structurally and functionally characterized. It consists of seven exons, spans 3107 base pairs, and its coding sequence initiates within exon 1. The primary structure of mouse rpL7 (270 amino acids), as inferred from the nucleotide sequence of the exons of the gene, and from the cDNA, is 12 residues longer than the rat counterpart. The rpL7 gene shares common structural features with most other mammalian ribosomal protein genes analyzed thus far. These include the lack of a canonical TATA box and a major transcription initiation site at a cytidine residue embedded in a stretch of 14 pyrimidines, flanked by C + G-rich regions. Transient expression assays revealed that the promoter region of rpL7 gene bears several regulatory elements, both upstream to the capsite and within the transcribed portion of the gene. One internal regulatory element was assigned to the first intron and a second one to a 20-base pair region spanning the first exon-intron junction. The activity of a deletion mutant of rpL32 gene, lacking its internal elements can be rescued by insertion, in the sense orientation, of the corresponding elements from the rpL7 gene. The unique spatial organization of the regulatory elements in rpL7 gene, as well as in other murine ribosomal protein genes examined thus far, might indicate that this common architecture is involved in the mechanism coordinating their expression.
A retrovirus packaging cell line was constructed by using portions of the Moloney murine leukemia virus in which the gag, pol, and env genes of the helper virus were separated onto two different plasmids and in which the psi packaging signal and 3' long terminal repeat were removed. The plasmid containing the gag and pol genes and the plasmid containing the env gene were cotransfected into NIH 3T3 cells. Clones that produced high levels of reverse transcriptase and env protein were tested for their ability to package the replication-defective retrovirus vectors delta neo and N2. One of the gag-pol and env clones (GP+E-86) was able to transfer G418 resistance to recipient cells at a titer of as high as 1.7 X 10(5) when it was used to package delta neo and as high as 4 X 10(6) when it was used to package N2. Supernatants of clones transfected with the intact parent gag-pol-env plasmid 3P0 had comparable titers (as high as 6.5 X 10(4) with delta neo; as high as 1.7 X 10(5) with N2). Tests for recombination events that might result in intact retrovirus showed no evidence for the generation of replication-competent virus. These results suggest that gag, pol, and env, when present on different plasmids, may provide an efficient and safe packaging line for use in retroviral gene transfer.
The most common translocation in human lymphoma, the t(14;18)(q32;q21), generates heterogeneous 4.2-7.2 kb Bcl-2-immunoglobulin (Ig) chimeric mRNAs resulting from alternative Bcl-2 5' exons and varied Ig 3' untranslated regions (UT). The normal human Bcl-2 gene has a three exon structure with an untranslated first exon, a facultative 220 bp intron I, but an enormous 370 kb intron II. S1 protection and primer extension analysis defined initiation sites in exon II associated with classic promoter elements and a decanucleotide (ATG-CAAAGCA) homologous with Ig variable region enhancers. Multiple initiation sites were also found in a GC-rich region with Sp1 binding motifs in exon I. Most t(14;18) breakpoints cluster within the 3' UT of Bcl-2 implicating that event in gene deregulation. The Bcl-2 gene introduced into the Ig constant (C gamma) locus of SU-DHL-6 displayed somatic mutation. While Bcl-2--Ig mRNAs demonstrated an unaltered 2.5 h half-life, the Bcl-2--Ig gene revealed an inappropriately high rate of transcription for a mature B-cell. This indicates the translocated Bcl-2 allele has escaped normal control mechanisms.
We examined the expression of the Bcl-2 gene at chromosome segment 18q21, that is translocated into the Ig heavy chain gene locus in t(14;18) bearing lymphomas. Bcl-2, while B cell associated, is expressed in a variety of hematopoietic lineages including T cells. Bcl-2 mRNA levels are high during pre-B cell development, the time at which the t(14;18) translocation occurs, but are down regulated with maturation. Like certain other oncogenes, Bcl-2 is quiescent in resting B cells but up-regulated with B cell activation. Mature B cell lymphomas with a t(14;18) have log-folds more mRNA than matched counterparts without the translocation. A sensitive S1 protection assay revealed that all transcripts in t(14;18) B cells were Bcl-2-Ig fusion mRNAs and originated from the translocated allele. Thus, there is a marked deregulation of Bcl-2 when it is introduced into the Ig locus in t(14;18) lymphomas.
The proto-oncogene c-fos is a major nuclear target for signal transduction pathways involved in the regulation of cell growth, differentiation, and transformation. Using the multistep skin carcinogenesis model, we have directly tested the ability of c-fos-deficient mice to develop cancer. Upon treatment with a tumor promoter, c-fos knockout mice carrying a v-H-ras transgene were able to develop benign tumors with similar kinetics and relative incidence as wild-type animals. However, c-fos-deficient papillomas quickly became very dry and hyperkeratinized, taking on an elongated, horny appearance. While wild-type papillomas eventually progressed into malignant tumors, c-fos-deficient tumors failed to undergo malignant conversion. Experiments in which v-H-ras-expressing keratinocytes were grafted onto nude mice suggest that c-fos-deficient cells have an intrinsic defect that hinders tumorigenesis. These results demonstrate that a member of the AP-1 family of transcription factors is required for the development of a malignant tumor.
The epithelial-specific integrin alpha 6 beta 4 is suprabasally expressed in benign skin tumors (papillomas) and is diffusely expressed in carcinomas associated with an increase in the proliferating compartment. Analysis of RNA samples by reverse transcriptase-PCR and DNA sequencing revealed that chemically or oncogenically induced papillomas (n = 8) expressed a single transcript of the alpha 6 subunit, identified as the alpha 6 A splice variant. In contrast, carcinomas (n = 13) expressed both alpha 6A and an alternatively spliced form, alpha 6B. Primary keratinocytes and a number of keratinocyte cell lines that vary in biological potential from normal skin, to benign papillomas, to well-differentiated slowly growing carcinomas exclusively expressed alpha 6A. However, I7, an oncogene-induced cell line that produces highly invasive carcinomas, expressed both alpha 6A and alpha 6B transcript and protein. The expression of alpha 6B in I7 cells was associated with increased attachment to a laminin matrix compared to cell lines exclusively expressing alpha 6A. Furthermore, introduction of an alpha 6B expression vector into a papilloma cell line expressing alpha 6A increased laminin attachment. When a papilloma cell line was converted to an invasive carcinoma by introduction of the v-fos oncogene, the malignant cells expressed both alpha 6A and alpha 6B, while the parent cell line and cells transduced with v-jun or c-myc, which retained the papilloma phenotype, expressed only alpha 6A. Comparative analysis of alpha 6B expression in cell lines and their derived tumors indicate that alpha 6B transcripts are more abundant in tumors than cell lines, and alpha 6B is expressed to a greater extent in poorly differentiated tumors. These results establish a link between malignant conversion and invasion of squamous tumor cells and the regulation of transcript processing of the alpha 6 beta 4 integrin.
Mouse tissue inhibitor of metalloproteinases-3 (mTIMP-3), a gene specifically not expressed in neoplastic JB6 cells, have been isolated recently through the use of the mRNA differential display technique (Sun, Y., Hegamyer, G., and Colburn, N. H. (1994) Cancer Res. 54, 1139-1144). We report here the full-length mTIMP-3 cDNA sequence, the promoter sequence and partial characterization, expression and induction of TIMP-3, and the possible molecular basis for the lack of mTIMP-3 expression in neoplastic JB6 cells. There are three transcripts arising from alternative polyadenylation of mouse TIMP-3 gene, having sizes of 4.6, 2.8, and 2.3 kilobase pairs, respectively. All three TIMP-3 transcripts are expressed in preneoplastic but not neoplastic JB6 cells. Computer analysis of cloned TIMP-3 promoter revealed six AP-1 binding sites, two NF-KB sites, a c-Myc site, and two copies of a p53 binding motif separated by eight base pairs with two mismatches at the second motif, along with many other cis elements. TIMP-3 gene expression was inducible by AP-1 and NF-KB activators, 12-O-tetradecanoylphorbol-13-acetate, and tumor necrosis factor-alpha only in preneoplastic cells with an induction peak at 2 h post-treatment, suggesting classification of mTIMP-3 as a member of the immediate early gene family. Southern blot, mutational analysis, and transient transcriptional activation experiments revealed that the lack of expression of mTIMP-3 in neoplastic JB6 cells was due neither to gross deletion nor to promoter mutation of the gene, nor was there a lack of transcription factors required for transcriptional activation. Instead, the lack of TIMP-3 expression in neoplastic JB6 cells may reflect an abnormal methylation of the gene. Both hyper- and hypomethylation of the mTIMP-3 gene are associated with complete down-regulation of gene expression in neoplastic JB6 cell lines. Treatment of neoplastic cells with the methylase inhibitor 5-azacytidine caused reexpression of the mTIMP-3 gene in a tumor cell line that showed hypermethylation but not in another that showed hypomethylation of the gene, suggesting a complex role for methylation in the silencing of gene expression.
The present study was directed to characterizing the reversion of neoplastic epidermal JB6 RT101 cells by AP-1 inhibiting
drugs. Treatment of tumorigenic JB6 RT101 cells with retinoic acid (RA), fluocinolone acetonide (FA) or forskolin (FN) induced
drug dependent (reversible) reversion of transformation. A synergistic effect on reversion was found with the three drugs
in combination. Cells reverted by these three drugs also showed reduced levels of AP-1 transcription factor activity. After
long term exposure of RT101 cells to FA, enrichment of flat revertants occurred in the population while a few unreverted cells
formed foci. These unreverted cells appeared to be FA-resistant. Cloning of cells following RA treatment revealed stable reversion
at least 2 months after drug withdrawal. Stable revertants showed lower basal AP-1 activity than RT101 cells (P < 0.01) and unstable revertants returned to transformed phenotype and elevated AP-1 activity within days following drug withdrawal.
To our knowledge this is the first demonstration that drug induced reversion co-selects for reduced AP-1 activity. These data
suggest that the JB6 RT101 cell line is a useful cell model for studying reversion of transformation and that inhibition of
AP-1 activity may be one molecular mechanism of reversion. Considering the development of resistance with FA alone and the
relative inefficiency of RA or FN alone, combinations of the three AP-1 activity inhibitors RA, FA and FN may be useful for
further animal and clinical studies.
Cell death by apoptosis plays a key role in skin development and homeostasis. Previous studies have shown that increased apoptosis characterizes several pathologic conditions affecting human skin. Thus, the pathogenesis of cutaneous diseases may involve an imbalance in the homeostatic mechanisms determining whether the death of keratinocytes will occur by terminal differentiation or apoptosis. We investigated the involvement of apoptosis in psoriasis. For this purpose, we assessed, in addition to morphology and DNA fragmentation, the expression of two putative apoptotic genes, bcl-2 and "tissue" transglutaminase, in normal and psoriatic skin. A large number of keratinocytes showing biochemical and morphologic features of cells undergoing apoptosis was observed in all the suprabasal layers of the psoriatic epidermis. The plaques from all patients analyzed showed a dramatic reduction in the number of bcl-2–positive cells localized in the basal cell compartment. In contrast, the psoriatic lesions presented a marked induction in "tissue" transglutaminase, which was localized specifically to the cytoplasm of apoptotic keratinocytes. "Tissue" transglutaminase protein staining was undetectable in normal epidermis. The bcl-2 and "tissue" transglutaminase staining pattern observed in psoriasis also was found in the skin of patients affected by lichen planus. These findings indicate that these two genes are regulated in an opposite fashion in psoriatic keratinocytes undergoing apoptosis, thus confirming their antithetic role in the cascade of events leading to the establishment of the mature apoptotic phenotype.Keywords: lichen planus, psoriasis, programmed cell death, melanocytes, DNA fragmentation
During human skin development, embryonic- and fetal-specific periderm cells and incompletely keratinized cells are replaced by keratinocytes that differentiate while stratifying to form the fully functional epidermis. Proliferating basal cells of fetal skin also develop into epidermal appendages such as hair follicles and glands. We demonstrate that programmed cell death, not emphasized in conventional epidermal biology, has an important function in establishing the final architecture of the human epidermis and its appendages. Immunohistochemical localization of transglutaminases in fetal periderm, intermediate epidermal cells, and within appendages coincides with DNA fragmentation indicating that apoptosis is involved in deletion of these stage-specific cells and remodeling of appendages. The data also suggest that terminal differentiation of epidermal cells might be a specialized form of apoptosis. The pattern of expression of bcl-2, a gene associated with survival of some cells, is exclusive of the distribution patterns of markers of the cell death pathway. Bcl-2 protein is correlated with specific morphogenetic events in hair follicles and eccrine sweat glands, and its presence in single cells of the hair follicle bulge suggests that Bcl-2 may be a stem cell marker. © 1994 Wiley-Liss, Inc.
Murine papilloma cell lines 308 and SP-1 have been used as recipients for transfected oncogenes to investigate malignant conversion. These cell lines express an activated crasHa gene with a codon 61 mutation and produce squamous papillomas when transplanted as skin grafts onto nude mice. They are not tumorigenic by subcutaneous injection. Both papilloma cell lines were stably transfected with plasmid DNA containing either a rearranged murine plasmacytoma-derived c-myc (minus exon 1), adenovirus 5 E1A, FBJ v-fos or a human c-fosl FBJ v-fos chimera, using cotransfection with the neomycin resistance gene contained in pSV2neo to select for transformants. Southern and northern blotting analysis confirmed the uptake and expression of exogenous DNA in both G418-selected cell lines and in the derived tumors. Unlike the E1A- and myc-containing plasmids, both fos constructs caused malignant conversion in either cell line, as defined by the squamous cell carcinoma histology of tumors from grafted cells and the development of carcinomas after subcutaneous injection into athymic nude mice. Immunofluorescence analysis for specific keratin gene expression indicated that tumors derived by introduction of either of the fos oncogenes were devoid of staining for K1, a 67 kDa epidermal keratin that is expressed in papillomas but not in squamous carcinomas. Tumors from E1A, myc, or pSV2neo transfectants expressed K1, although in a focal distribution. The malignant phenotype induced by the fos oncogene constructs was not associated with the ability to form agar colonies in vitro or to express -glutamyl transpeptidase in the tumors. Since both 308 and SP-1 were sensitive to the fos oncogene for malignant conversion and insensitive to E1A or myc, it is possible that fos may cooperate with the endogenous-activated c-rasHa gene to convert these cells to malignancy. However, since -glutamyl transpeptidase activity is found in the majority of chemically induced mouse skin carcinomas that possess an activated crasHa gene, fos activation may not be a common pathway for spontaneous malignant conversion.
Using the technique of differential display of mRNAs, we isolated 42 differentially expressed cDNAs from squamous papillomas induced in the skin of SENCAR mice by a standard 7,12-dimethylbenz(a)anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate promotion protocol. Thirty-one of the cDNAs were cloned and sequenced and the sequences compared with the catalogued sequences in the GenBank/EMBL DNA databases. Two of the cDNA clones-pJTM-4 and pJTM-12-showed insignificant homology with the known murine DNA and protein sequences and thus are novel gene transcripts. Putative protein product of the cDNA clone pJTM-15 showed homology to a stretch of amino acids (with a difference of two amino acids out of 16 compared) within the coding region of the previously reported murine platelet derived growth factor inducible KC protein cDNA (KC/Gro, a member of small cytokine family). By Northern blot analysis, transcripts of pJTM-4 and pJTM-12 cDNAs were found to be expressed in mRNA samples prepared from similarly induced skin tumors in other mice but were not expressed in the epidermis of either untreated mice or in uninvolved epidermis of the tumor bearing mice. Expression of pJTM-4 and pJTM-12 was also detected in mouse sarcoma and lymphosarcoma cell lines but not in a melanoma cell line. These results suggest possible involvement of these novel genes in non-melanoma skin cancer. Full length sequences of these novel transcripts and characterization of their protein products may provide useful information regarding the molecular events which occur during multistage skin carcinogenesis.
The significance of apoptosis in relation to the development and progression of prostate cancer remains largely undefined. bcl-2 is an oncogene that functions by overriding apoptosis. bcl-2 expression was localized to the basal epithelial cells in the normal human prostate with the use of immunohistochemistry. Androgen-dependent and androgen-independent prostate carcinomas were evaluated immunohistochemically for bcl-2 expression. bcl-2 was undetectable in 13 of 19 cases of androgen-dependent cancers. In contrast, androgen-independent cancers displayed diffuse, high levels of bcl-2 staining (P < 0.01). In rats, steady-state levels of bcl-2 mRNA, assessed by S1 assays, reached maximum levels 10 days following castration. Addition of exogenous testosterone with, or without, flutamide demonstrated that the increased bcl-2 mRNA resulted from androgen ablation. Our findings indicate that bcl-2 expression is augmented following androgen ablation and is correlated with the progression of prostate cancer from androgen dependence to androgen independence.
When messenger RNA precursors (pre-mRNAs) containing alternative 5' splice sites are spliced in vitro, the relative concentrations of the heterogeneous ribonucleoprotein (hnRNP) A1 and the essential splicing factor SF2 precisely determine which 5' splice site is selected. In general, an excess of hnRNP A1 favors distal 5' splice sites, whereas an excess of SF2 results in utilization of proximal 5' splice sites. The regulation of these antagonistic activities may play an important role in the tissue-specific and developmental control of gene expression by alternative splicing.
Previous studies have implicated the rasHa oncogene in the initiation of skin carcinogenesis and the fos oncogene in malignant progression of premalignant skin cell lines. To determine if these two oncogenes are sufficient to convert normal keratinocytes to cancer cells, freshly isolated mouse keratinocytes were coinfected with replication-defective (psi-2) v-rasHa and v-fos viruses in culture. When tested in nude mice within several days of infection, v-fos/v-rasHa-coinfected keratinocytes produced squamous cell carcinomas. Introduction of v-fos alone resulted in normal or hyperplastic skin, whereas v-rasHa alone produced squamous papillomas. These results indicate that two oncogenes are sufficient to produce the malignant phenotype in epidermal cells. Furthermore, they clearly link the fos oncogene with malignant conversion. Since fos acts as a transcriptional regulator of other genes, malignant conversion may be an indirect consequence of the overexpression of the fos-encoded protein leading to a change in the expression of fos-controlled cellular genes.
The t(14; 18) chromosomal translocation of human follicular B-cell lymphoma juxtaposes the bcl-2 gene with the immunoglobulin heavy chain locus. The bcl-2 immunoglobulin fusion gene is markedly deregulated resulting in inappropriately elevated levels of bcl-2 RNA and protein. Transgenic mice bearing a bcl-2 immunoglobulin minigene demonstrate a polyclonal expansion of resting yet responsive IgM-IgD B cells which display prolonged cell survival but no increase in cell cycling. Moreover, deregulated bcl-2 extends the survival of certain haematopoietic cell lines following growth-factor deprivation. By using immunolocalization studies we now demonstrate that Bcl-2 is an integral inner mitochondrial membrane protein of relative molecular mass 25,000 (25k). Overexpression of Bcl-2 blocks the apoptotic death of a pro-B-lymphocyte cell line. Thus, Bcl-2 is unique among proto-oncogenes, being localized to mitochondria and interfering with programmed cell death independent of promoting cell division.
SF2 is a 33 kd protein factor required for 5' splice site cleavage and lariat formation during pre-mRNA splicing in HeLa cell extracts. In addition to its essential role in constitutive splicing, SF2 can strongly influence 5' splice site selection. When pre-mRNAs containing multiple cis-competing 5' splice sites are spliced in vitro, high concentrations of purified SF2 promote the use of the 5' splice site closest to the 3' splice site. However, SF2 discriminates properly between authentic and cryptic splice sites. These effects of SF2 on splice site selection may reflect the cellular mechanisms that prevent exon skipping and ensure the accuracy of splicing. In addition, alterations in the concentration or activity of SF2, and of other general splicing factors, may serve to regulate alternative splicing in vivo.
Papillomas induced by standard initiation-promotion protocols progress to carcinomas at a low frequency. Experimental protocols were developed to elicit papillomas with a higher probability of malignant conversion. SENCAR mice initiated by 7,12-dimethylbenz[a]anthracene were promoted by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 5, 10, 20 or 40 weeks. With promotion for 10 weeks or more, a peak of papilloma incidence at 16-20 weeks was followed by a 35-40% decrease within 3 months. A much lower papilloma response was seen in mice promoted for 5 weeks, but these papillomas persisted. The yield of malignant tumors was similar in all four groups, with 20-25 carcinomas per group of 30 mice. Thus, the papillomas induced by the first few TPA treatments are much more likely to progress to carcinomas than those which appear later. In a separate study, initiated Charles River CD-1 mice were promoted with TPA for either 12 or 52 weeks. Acetone solvent treatment was begun at Week 13 in the group treated 12 weeks with TPA. At Week 16, the papilloma incidence was identical in the two groups of mice. However, by Week 28, the papilloma yield in the continuous TPA group had increased and was twice that of the acetone group, in which papillomas had regressed. The first carcinoma arose 14 weeks earlier with continuous TPA, but the final number of carcinomas per group of 40 mice was 17 with TPA and 20 with acetone. Neither the increase in papillomas in TPA-treated mice nor the regression of papillomas after cessation of promotion with TPA affected the final carcinoma yield. This result suggests that TPA-dependent papillomas are very unlikely to progress to carcinomas. In a third experiment, promotion of initiated SENCAR mice with mezerein resulted in a small number of papillomas which had a much higher probability of progression to carcinomas than the large number of papillomas promoted by TPA. The ability to induce papillomas promoted by TPA. The ability to induce papillomas with a known probability of conversion to carcinomas will facilitate the identification of markers associated with malignant progression.
Studies of the mutagenic action required for specific chemicals to produce benign or malignant tumours suggest that in mouse skin at least two genetic events occur before carcinoma formation. The isolation of an activated form of the c-rasH gene from skin papillomas has provided evidence that this gene may be a target for the first mutation, which could constitute the initiating mutation in skin carcinogenesis. In vitro studies indicate that the v-rasH gene of Harvey murine sarcoma virus (Ha-MSV), a replication-defective transforming retrovirus, could impart a conditional initiated phenotype on cultured keratinocytes by blocking their ability to differentiate terminally and arresting them in a late basal cell stage of maturation. We now show that when the Ha-MSV v-rasH gene is introduced into cultured keratinocytes by a defective retroviral vector, skin grafts constructed with cells carrying the mutated ras oncogene produce papillomas on athymic nude mouse recipients. Furthermore, the expression of the exogenous oncogene seems to be regulated at the transcriptional level in the differentiated portions of the benign tumour.
We describe a set of murine retrovirus-based vectors that include unique cloning sites for insertion of cDNAs such that the cDNA can be driven by either the retroviral long terminal repeat, the immediate early promoter of human cytomegalovirus, or the simian virus 40 early promoter. The vectors carry the neomycin phosphotransferase gene expressed from an alternate promoter as a selectable marker. These vectors have been constructed to prevent viral protein synthesis from the remaining viral sequences, to yield high-titer virus stocks after introduction into retrovirus packaging cells, and to eliminate homologous overlap with viral DNAs present in retrovirus packaging cells in order to prevent helper virus production. Methods for generating high-titer virus are described.
We have identified a 24-kilodalton protein that is the product of the human bcl-2 gene, implicated as an oncogene because of its presence at the site of t(14;18) translocation breakpoints. The Bcl-2 protein was detected by specific, highly sensitive rabbit antibodies and was shown to be present in a number of human lymphoid cell lines and tissues, as well as in mouse B cells transfected with a bcl-2 cDNA construct. Characterization of the Bcl-2 protein demonstrated that it has a lipophilic nature and is associated with membrane structures, probably by means of its hydrophobic carboxy-terminal membrane-spanning domain. In t(14;18)-carrying cell lines, the protein is predominantly localized to the perinuclear endoplasmic reticulum, with a minor fraction in the plasma membrane. These properties, together with the observations that Bcl-2 does not have a characteristic signal peptide and is not glycosylated, suggest that it is an integral-membrane protein that spans the bilayer at its C-terminal hydrophobic region but is exposed only at the cytoplasmic surface. The relative abundance of the Bcl-2 protein in various human lymphoid cell lines correlated with transcription of the bcl-2 gene. The protein was abundant in all t(14;18)-carrying cell lines and lymphomas and was also found at lower levels in pre-B-cell lines and nonmalignant lymphoid tissues that do not carry t(14;18) translocations. These results suggest that the Bcl-2 protein is functional in normal B lymphocytes and that a quantitative difference in its expression may play a role in the pathogenesis of lymphomas carrying the t(14;18) translocation.
cDNAs for the bcl-2 mRNA were cloned from a human common acute lymphoblastic leukemia cell line. Nucleotide sequence analyses showed that the 6 kb bcl-2 mRNA potentially encodes a 26 kd protein that is homologous to a predicted Epstein-Barr virus protein. Most t(14;18) translocation breakpoints cluster within a narrow region of a 5.4 kb exon that contains a long 3'-untranslated region of the bcl-2 mRNA. As a result of t(14;18) translocation, hybrid bcl-2/immunoglobulin heavy chain transcripts are produced that consist of the 5' half of the bcl-2 mRNA fused to a "decapitated" immunoglobulin heavy chain mRNA. Nucleotide sequence analyses confirmed that the hybrid transcripts continue to encode a normal bcl-2 protein. Our results suggest that t(14;18) translocations alter expression of the bcl-2 gene both by transcriptional activation and by abnormal posttranscriptional regulation of bcl-2 mRNA.
We have cloned the mouse bcl-2 (mbcl-2) genomic locus and analyzed it in detail. The gene is comprised of two exons separated by more than 15 kb. Two species of mRNAs are produced, and DNA sequencing analysis shows that they code for two proteins differing at their C terminus: a 7.5 kb transcript codes for a polypeptide of 236 amino acids, mbcl-2 alpha, and a 2.4 kb transcript, which derives from the 5' exon only, codes for a protein of 199 amino acids, mbcl-2 beta. The gene is characterized by very long (5' about 1.4 kb, and 3' about 5.1 kb) untranslated regions surrounding the relatively short coding region. We have mapped the 5' end of the mbcl-2 mRNAs by S1 protection analysis, and we have analyzed the promoter region. The expression of the mbcl-2 gene was analyzed in different cell lines and in mouse tissues. Expression is tissue-specific in adult tissues: spleen and thymus express the highest level of mbcl-2 transcripts. The mbcl-2 gene maps to mouse chromosome 1.
We have developed four murine epidermal cell lines which form squamous papillomas when grafted to athymic nude mice in a reconstituted skin. Two of the lines, SP-1 and BP-4, were derived from pools of papillomas produced on SENCAR and BALB/c mouse skin, respectively, by initiation with 7,12-dimethylbenz(a)anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate. Line 308 was derived from BALB/c mouse skin initiated in vivo with 7,12-dimethylbenz(a)anthracene, culture of the epidermal cells, and selection of cells resistant to Ca2+-induced terminal differentiation. Line LC14 was derived from untreated, cultured newborn BALB/c mouse primary epidermal cells which spontaneously developed resistance to Ca2+-induced terminal differentiation. Each line has an activated rasHa gene with a mutation within codon 61. Cells from all four lines, in contrast to normal primary epidermal cells, survive in medium with Ca2+ levels greater than 0.1 mM. Clonal growth studies in culture showed a unique growth pattern for each of the four lines in medium with 1.4 mM and 0.05 mM Ca2+, with or without 12-O-tetradecanoylphorbol-13-acetate. Early passage cells of these lines should provide a valuable resource for detecting genes or genetic alterations which complement an activated ras gene to cause malignant conversion and for studying the biology of tumor promotion.
A common feature of follicular lymphoma, the most prevalent haematological malignancy in humans, is a chromosome translocation (t(14;18] that has coupled the immunoglobulin heavy chain locus to a chromosome 18 gene denoted bcl-2. By analogy with the translocated c-myc oncogene in other B-lymphoid tumours bcl-2 is a candidate oncogene, but no biological effects of bcl-2 have yet been reported. To test whether bcl-2 influences the growth of haematopoietic cells, either alone or together with a deregulated c-myc gene, we have introduced a human bcl-2 complementary DNA using a retroviral vector into bone marrow cells from either normal or E mu-myc transgenic mice, in which B-lineage cells constitutively express the c-myc gene. Bcl-2 cooperated with c-myc to promote proliferation of B-cell precursors, some of which became tumorigenic. To determine how bcl-2 expression impinges on growth factor requirements, the gene was introduced into a lymphoid and a myeloid cell line that require interleukin 3 (IL-3). In the absence of IL-3, bcl-2 promoted the survival of the infected cells but they persisted in a G0 state, rather than proliferating. These results argue that bcl-2 provided a distinct survival signal to the cell and may contribute to neoplasia by allowing a clone to persist until other oncogenes, such as c-myc, become activated.
From an acute B-cell leukemia cell line, a DNA probe was obtained that was specific for chromosome 18 and flanked the heavy
chain joining region of the immunoglobulin heavy chain locus on chromosome 14. This probe detected rearrangement of the homologous
DNA segment in the leukemic cells and in follicular lymphoma cells with the t(14:18) chromosome translocation but not in other
neoplastic or normal B or T cells. The probe appears to identify bcl-2, a gene locus on chromosome 18 (band q21) that is unrelated
to known oncogenes and may be important in the pathogenesis of B-cell neoplasms with this translocation.
Modification of the ionic calcium concentration in the culture medium markedly alters the pattern of proliferation and differentiation in cultured mouse epidermal cells. When medium calcium is lowered to 0.05--0.1 mM, keratinocytes proliferate rapidly with a high growth fraction and do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured and cloned in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces, abundant microvilli and perinuclear organization of tonofilaments and cellular organelles. Desmosomes are absent. Epidermal cells growing as a monolayer in low Ca++ can be induced to terminally differentiate by adding calcium to the level normally found in the culture medium (1.2 mM). Cell-to-cell contact occurs rapidly and desmosomes form within 2 hr. The cells stratify by 1--2 days and terminally differentiate with cell sloughing by 3--4 days. After Ca++ addition, DNA synthesis decreases with a lag of 5--10 hr and is totally inhibited within 34 hr. In contrast, RNA and protein synthesis continue at 40--50% of the low Ca++ level at day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch.
Primary mouse epidermal cells underwent spontaneous malignant transformation in culture. TWelve malignant epidermal cell lines were established which produced squamous cell carcinomas in syngeneic hosts. These lines were used to define criteria for recognizing transformed epidermal cells in vitro. Growth in suspension in agar, agarose, or Methocel was minimal for 11 of the lines. All lines tested retained specific epidermal antigens (pemphigus, pemphigoid, keratin) by indirect immunofluorescence, but keratin content was reduced when quantified by radioimmunoassay. Basal activity of ornithine decarboxylase and activity induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate were variable among lines. All malignant lines as well as normal epidermal cells grew well at reduced extracellular calcium concentrations. When the extracellular calcium was elevated, normal cells ceased proliferation, terminally differentiated, and sloughed from the culture dish, while malignant cells continued to proliferate although they expressed differentiative functions. These results indicate that malignant transformation in epidermis is associated with a fundamental alteration in the program of terminal differentiation which allows some cells to escape the proliferative block and cell death which accompanies differentiation in normal keratinocytes. This alteration should be useful to select for transformants during the process of carcinogenesis in vitro.
ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.
During human skin development, embryonic- and fetal-specific periderm cells and incompletely keratinized cells are replaced by keratinocytes that differentiate while stratifying to form the fully functional epidermis. Proliferating basal cells of fetal skin also develop into epidermal appendages such as hair follicles and glands. We demonstrate that programmed cell death, not emphasized in conventional epidermal biology, has an important function in establishing the final architecture of the human epidermis and its appendages. Immunohistochemical localization of transglutaminases in fetal periderm, intermediate epidermal cells, and within appendages coincides with DNA fragmentation indicating that apoptosis is involved in deletion of these stage-specific cells and remodeling of appendages. The data also suggest that terminal differentiation of epidermal cells might be a specialized form of apoptosis. The pattern of expression of bcl-2, a gene associated with survival of some cells, is exclusive of the distribution patterns of markers of the cell death pathway. Bcl-2 protein is correlated with specific morphogenetic events in hair follicles and eccrine sweat glands, and its presence in single cells of the hair follicle bulge suggests that Bcl-2 may be a stem cell marker.
The recently described mRNA differential display method provides an attractive tool for the isolation of genes showing regulated expression in a variety of systems. A key step in this technique consists of the isolation of PCR-synthesized radioactive cDNAs corresponding to differentially expressed mRNAs. Here, we show that the purified cDNAs remain contaminated with unrelated cDNA sequences that may lead to the artifactual isolation of false positives in the subsequent steps of the method. A powerful assay for the detection and elimination of this contaminating material, allowing the specific isolation of clones corresponding to the regulated genes identified by the differential display, is provided.
Transgenic mice that expressed v-fos exclusively in the epidermis by means of a human keratin K1-based targeting vector (HK1.fos) developed preneoplastic epidermal hyperplasia and hyperkeratosis after long latency and an associated wound promotion stimulus. To assess the requirements for papilloma formation and malignant conversion and determine the sensitivity to a chemical promotion stimulus, HK1.fos mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). HK1.fos mice were sensitive to TPA promotion but developed papillomas only after long latency (20-30 weeks of promotion) and in relatively few numbers per animal, suggesting the necessity of an additional genetic event prior to overt lesion formation. Consistent with this idea, at 60 weeks, on cessation of TPA promotion, these HK1.fos TPA-papillomas were found to be autonomous, TPA-independent tumors which persisted, grew larger, and converted to malignancy. Analysis of HK1.fos tumor RNA and DNA identified endogenous c-rasHa mutations at codons 12 and 61 in papillomas and carcinomas; however, no p53 tumor suppressor gene mutations were detected. These data indicate that epidermal expression of v-fos induces sensitivity to TPA promotion, but since additional genetic events, such as endogenous c-rasHa activation, appear to be required in tumorigenesis, v-fos may predominantly play a role in the mechanism of promotion to achieve papilloma autonomy and TPA independence. Furthermore, spontaneous malignant conversion in this model does not appear to involve mutations in the p53 tumor suppressor gene.
Differential display and RNA arbitrary primed polmerase chain reaction are methods recently designed to identify and isolate differentially expressed genes. Methodological modifications have since been introduced to streamline the techniques. The major effort has centered on how to eliminate false positives as approached from a variety of angles, ranging from RNA sample preparation, northern blot confirmation, primer length variation, to better experimental design.
The mts1 gene is one of the genes specifically expressed in mouse metastatic tumors and tumor cell lines. In this paper, we present data on cloning and sequencing of two variants of human mts1 cDNAs (hu-mts1 and hu-mts1 (var)), as well as of the corresponding region in the human genome. Comparison of the genomic sequence with the sequence of the mts1 cDNAs demonstrates presence of two alternatively spliced variants of the mts1 in the human osteosarcoma cell line (OHS). The alternative splicing occurs within the 5'-untranslated region (UTR) of human mts1 pre-mRNA. Both splice variants, hu-mts1 and hu-mts1 (var), retain similar stability in the cells, contain one open reading frame coding for the MTS1 protein and differ only slightly in their translational capacity. The splice variants demonstrate dramatic variations in the level of expression in different human tissues and in human tumor cell lines. Although we have not revealed substantial differences in the mode of action of the two splice variants in the cells, the observed tissue specificity of expression supports the notion that it plays an important role in determining the activity of mts1 in different tissues.
Transgenic mice have been previously established that express v-rasHa or v-fos exclusively in the epidermis by means of a targeting vector based on the human keratin 1 gene (HK1). Epidermal expression of v-rasHa (HK1.ras) or v-fos (HK1.fos) resulted in hyperplasia, hyperkeratosis, and later, in benign tumors. To assess the potential for oncogene cooperation in vivo mating experiments were performed. Resultant HK1.fos/ras mice exhibited an obvious increase in the severity of neonatal and juvenile preneoplastic phenotypes, together with the immediate onset of tumorigenesis as compared to single oncogene sibling controls. The HK1.fos/ras tumors grew aggressively and often compromised the animals by 10-12 weeks. However, tumors remained benign as determined by histotype and specific keratin markers. These data indicate that v-fos can cooperate with an initiating v-rasHa phenotype to generate autonomous papillomas, but additional events are required for malignant conversion.
The present study was directed to characterizing the reversion of neoplastic epidermal JB6 RT101 cells by AP-1 inhibiting drugs. Treatment of tumorigenic JB6 RT101 cells with retinoic acid (RA), fluocinolone acetonide (FA) or forskolin (FN) induced drug dependent (reversible) reversion of transformation. A synergistic effect on reversion was found with the three drugs in combination. Cells reverted by these three drugs also showed reduced levels of AP-1 transcription factor activity. After long term exposure of RT101 cells to FA, enrichment of flat revertants occurred in the population while a few unreverted cells formed foci. These unreverted cells appeared to be FA-resistant. Cloning of cells following RA treatment revealed stable reversion at least 2 months after drug withdrawal. Stable revertants showed lower basal AP-1 activity than RT101 cells (P < 0.01) and unstable revertants returned to transformed phenotype and elevated AP-1 activity within days following drug withdrawal. To our knowledge this is the first demonstration that drug induced reversion co-selects for reduced AP-1 activity. These data suggest that the JB6 RT101 cell line is a useful cell model for studying reversion of transformation and that inhibition of AP-1 activity may be one molecular mechanism of reversion. Considering the development of resistance with FA alone and the relative inefficiency of RA or FN alone, combinations of the three AP-1 activity inhibitors RA, FA and FN may be useful for further animal and clinical studies.
Programmed cell death is a controlled process that leads to the elimination of single cells via apoptosis, a mode of cell death with a characteristic morphology. During epidermal differentiation, keratinocytes migrate outward to become terminally differentiated cornified cells in a process involving programmed cell death pathway(s) and apoptosis. The molecular mechanisms regulating epidermal differentiation and apoptosis have not yet been elucidated. Here we show that a mouse keratinocyte cell line, Pam212, undergoes spontaneous apoptosis in culture. Apoptosis of Pam212 cells is demonstrated by both morphology and DNA oligonucleosomal degradation. The expression of bcl-2, a gene implicated in the negative control of apoptosis, was down-regulated in these cells by transfecting a bcl-2-antisense expression vector. The cells that down-regulate bcl-2 expression exhibit enhanced apoptosis and further progress in the epidermal differentiation pathway. We analyzed the expression patterns of several genes that have been implicated in apoptosis in other systems. We show that the mRNA levels of c-myc, c-myb, c-fos, tumor necrosis factors (TNF) alpha and beta, TNF receptors I and II, interleukin 1 alpha, IFN-gamma, and transforming growth factor beta increase in the antisense-transfected cells. We suggest that bcl-2 influences epidermal differentiation in Pam212 keratinocyte cells, and maybe in vivo, by negatively regulating several genes that are involved in apoptosis.
Apoptosis or programmed cell death represents a mechanism by which cells possessing DNA damage can be deleted. The bcl-2 proto-oncogene is a known inhibitor of apoptosis that may allow the accumulation and propagation of cells containing genetic alterations. To determine if and when the bcl-2 gene is activated during colorectal tumorigenesis and its relationship to p53, we analyzed normal mucosa, hyperplastic and dysplastic epithelial polyps, and carcinomas for the expression of these markers using immunohistochemistry. Whereas bcl-2 staining was restricted to basal epithelial cells in normal and hyperplastic mucosa, bcl-2 expression was detected in parabasal and superficial regions in dysplastic polyps and carcinomas. An inverse correlation was found between bcl-2 and p53 expression in adenomas, suggesting that these markers may regulate a common cell death pathway. Furthermore, carcinomas with a high percentage of bcl-2-positive cells were significantly more likely to have low rates of spontaneous apoptosis, as determined histologically, than those cancers with low or absent bcl-2 expression. Abnormal activation of the bcl-2 gene appears to be an early event in colorectal tumorigenesis that can inhibit apoptosis in vivo and may facilitate tumor progression.
The bcl-2 proto-oncogene encodes a protein that protects cells from programmed cell death (apoptosis). High levels of this protein confer a growth advantage to neoplastic cells even in the absence of a high mitotic rate. This gene is involved in the interchromosomal 14;18 translocation, an abnormality present in more than two-thirds of follicular lymphomas and in about 25% of other non-Hodgkin's lymphomas of the lymph nodes. A recent study also demonstrated the presence of high levels of bcl-2 protein in solid tumors such as squamous cell carcinomas of the lung and related it to a better prognosis. We analyzed bcl-2 protein expression in 20 cases each of basal- and squamous-cell carcinoma and in 5 biopsy specimens of normal skin, using a monoclonal anti-bcl-2 protein antibody with a standard 3-step immunoperoxidase technique on routinely fixed, paraffin-embedded tissue sections. Normal skin showed positive staining of the majority of keratinocytes in the epidermal basal layer. Bcl-2 positivity was also observed within the outer root sheath and the mesenchymal cells of the follicular papillae, the clear cells of the eccrine glands, and in some melanocytes at the dermo-epidermal junction. Neoplastic cells in all cases of basal-cell carcinoma showed a positive cytoplasmic reaction for bcl-2. All biopsy specimens of squamous-cell carcinoma were negative. Expression of bcl-2 protein could also be observed in the majority of peri- and intratumoral lymphocytes in both basal-cell carcinoma and squamous-cell carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)
The hair follicle undergoes a cycle of growing, regressing, and resting phases (anagen, catagen, telogen, respectively). As the follicle enters catagen, the cells of the lower, cycling portion undergo a process of controlled cell death (apoptosis). Understanding the mechanism of apoptosis in the follicle should give insight into one of the control steps of hair cycling. In this study we sought the expression of bcl-2, a protooncogene associated with apoptosis control, in the cycling follicle of the adult mouse. Using a monoclonal antibody to the mouse protein we immunolocalized bcl-2 gene product in the cycling pelage follicle of the C57/B6 adult mouse. The protein was expressed in the follicular papilla (a non-cycling portion of the follicle) throughout the cycle-including telogen. The cycling follicular epithelium, however, showed positive antibody staining in anagen, which decreased in catagen and disappeared in telogen. In anagen the cells of the bulb, bulge, and basal layer of the outer root sheath expressed the bcl-2 protein. Understanding the action of this apoptosis-inhibiting molecule should serve to elucidate the dynamics of follicular cycling.
Trichoepitheliomas (TE) are benign follicular neoplasms which are frequently confused with basal cell carcinomas (BCC). It is important to distinguish these entities precisely, as the treatments and prognoses are different. To this end, we stained a series of formalin-fixed, paraffin-embedded specimens of unequivocal TE and BCC with antibodies directed against bcl-2, an oncogene associated with programmed cell death, and known to be overexpressed in some malignant tumours. The TE showed staining of tumour cells limited to the outermost layer of the proliferation. The BCC tumour cells demonstrated diffuse staining throughout the tumour nodules. This difference in staining pattern was then applied to more equivocal cases, and seemed clearly to separate the entities. The observed findings may prove to be of diagnostic help in distinguishing borderline cases, and also offer some possible explanations for the biological differences between these neoplasms.
A follicular origin for some skin tumors has been hypothesized in both humans and animal models. Because of its rapid and sensitive response to tumor promoter treatment, a v-Ha-ras transgenic (TG.AC) mouse line was used to determine the origins of epidermal papillomas. Using histological studies and transgene expression as a marker for papilloma development, we determined that pedunculated papillomas arose from focal hyperplasias of the permanent portion of the follicular epithelium in phorbol 12-myristate 13-acetate-treated TG.AC mouse skin. Damage to the hair follicle by depilation was also sufficient to induce papillomas that were histologically indistinguishable from those produced by chemical exposure. Identification of the cellular origins of papillomas in this transgenic mouse model will allow for an analysis of the role of the hair follicle and hair cycle-associated signaling in tumor development.
The loss of heterozygosity (LOH) at the bcl-2 gene locus and the expression of the bcl-2 gene were examined in gastric and colorectal carcinoma cell lines and carcinoma tissues. LOH at the bcl-2 locus was detected in 24% (4/17) of gastric and 60% (6/10) of colonic carcinomas, all of which were well differentiated adenocarcinomas, whereas LOH was not seen in poorly differentiated ones. On the other hand, 24% (5/21) of poorly differentiated stomach cancers overexpressed bcl-2 gene, whereas no overexpression was detected in well differentiated stomach cancer. Three gastric and three colorectal carcinoma cell lines, all of which were derived from poorly differentiated adenocarcinomas, expressed considerable levels of bcl-2 mRNA and protein. These results suggest that LOH at the bcl-2 locus is frequently associated with well differentiated adenocarcinomas of the stomach and colon, and bcl-2 overexpression has implications for the development of poorly differentiated adenocarcinomas of the gastrointestinal tract.