Dual role of the activated epidermal growth factor receptor in renal tubular cells during stress.

The paper by Sarro et al. raises the therapeutically important possibility that activation of the epidermal growth factor receptor (EGFR) after proximal tubule injury serves an unexpected death function and the EGFR-mediated survival signaling can be restored by identification of parallel and interacting pathways.

STAT3 attenuates EGFR-mediated ERK activation and cell survival during oxidant stress in mouse proximal tubular cells.

We have shown that renal epithelial cell survival depends on the sustained activation of the extracellular signal-regulated protein kinase (ERK) and lack of this activation was associated with death during oxidative stress. ERK is activated via the canonical epidermal growth factor receptor (EGFR)-Ras-MEK pathway, which could be attenuated by oxidants. We now show that the failure to activate ERK in a sustained manner during severe oxidative stress is owing to the activation of the signal transducer and activator of transcription-3 (STAT3) rather than the failure to activate the EGFR. Tyrosine phosphorylation of the EGFR and STAT3 was studied in hydrogen peroxide (H(2)O(2))-treated mouse proximal tubule (TKPTS) cells or in mouse kidney after ischemia/reperfusion (I/R) injury by Western blotting. STAT3 activation was inhibited by either pharmacologically (AG490) through its upstream janus kinase (JAK2) or by a dominant-negative STAT3 adenovirus. EGFR was inhibited by AG1478. Survival was determined by fluorescence-activated cell sorter analysis and trypan blue exclusion. We found that the EGFR was phosphorylated on its major autophosphorylation site (Tyr1173) regardless of the H(2)O(2) dose. On the other hand, both I/R and severe oxidative stress - but not moderate stress - increased tyrosine phosphorylation of STAT3 in an EGFR and JAK2-dependent manner. Inhibition of JAK2 or STAT3 lead to increased ERK activation and survival of TKPTS cells during severe oxidative stress. Our data suggest a role of tyrosine-phosphorylated STAT3 in the suppression of ERK activation. These data suggest that the STAT3 pathway might represent a new target for improved survival of proximal tubule cells exposed to severe oxidant injury.

Early effects of transplatin on oncogene activation in vivo.

The aim of the study was to investigate the early effect of Transplatin (the stereo-isomer of Cisplatin) on oncogenes in inbred CBA/Ca mice. Cisplatin is commonly used for the treatment of squamous cell carcinomas of the head and neck. Cisplatin has a strong oncogene activation effect compared to the structural analogue Transplatin. Body weight equivalent amounts of a human dose of Transplatin were administered intra-peritoneally to 6- to 8-week-old, inbred, female CBA/Ca mice. Twenty-four, 48 and 72 hours after the treatment, RNA was isolated from the target organs and the expressions of c-myc, Ha-ras and p53 genes were examined. Investigation of early changes showed no significant overexpression compared to Cisplatin, which had a significant effect on oncogene expression in the "short-term" in vivo test system.

Early effects of different cytostatic protocols for head and neck cancer on oncogene activation in animal experiments.

In vivo investigations on oncogenes and onco-suppressor genes may provide new findings on the potential carcinogenic effects of various cytostatic protocols inducing secondary tumours of the head and neck. Further surgeries are often necessary due to regional recurrence after the Cisplatin-supplemented BVM (Bleomycin, Vincristine, Methotrexate) protocol in the treatment of human head and neck tumours. Our earlier studies have illustrated the carcinogenic and mutagenic potential of Cisplatin. The effect of Cisplatin on the alteration of different onco- and suppressor genes has also been proven. Our present study aimed at investigating the early effects of the BVM and the CFu (Cisplatin, 5-Fluorouracil) protocols on early oncogene and tumour suppressor gene expressions in mice. Body weight equivalent amounts of cytostatics were administered intraperitoneally to 6- to 8-week-old, inbred, female CBA/Ca mice. Twenty-four, 48 and 72 hours after the treatment, RNA was isolated from the target organs and the quantitative expression of c-myc, Ha-ras and p53 genes were examined. The protocols caused detectable changes. A "short-term" in vivo test, the 24-hour examination of gene expression, is suitable for detecting early effects of carcinogen exposure. The alterations of gene expression, caused by the Cisplatin-containing protocol, draw attention to the probable role of Cisplatin in the development of regional recurrence and to the possibility of prevention.

Clinical and molecular responses in high-grade intraepithelial neoplasia treated with topical imiquimod 5%.

OBJECTIVE: To assess the clinical and molecular response of patients with recurrent high-grade vulvar, vaginal, or cervical intraepithelial neoplasia treated with topical 1-2(2-methylpropyl)-1H-imidazo [4,5-c] quinolin-4-amine (imiquimod) cream 5%, an immune response modifier with known efficacy in the treatment of external genital warts. METHODS: This is the first case series in the peer-reviewed literature reporting the use of imiquimod in high-grade intraepithelial neoplasia of the lower genital tract. Eight patients with high-grade intraepithelial neoplasia were treated with imiquimod in the gynecological oncology clinic and the HIV gynecology clinic at The University of Texas Medical Branch at Galveston. Frozen biopsies were available for RNA extraction on four patients before and after therapy. Using semiquantitative reverse transcription-PCR, we measured RNA levels of IFNs alpha and gamma, 2',5'-oligoadenylate synthetase, as well as CD4 and CD8 lymphocyte markers. RESULTS: Of the patients treated, four had complete responses, two had partial responses, one progressed, and one did not tolerate the therapy. Of the four complete responders, two remained disease-free (mean follow-up, 33 months). 2',5'-Oligoadenylate synthetase RNA expression showed an increased trend after therapy. CONCLUSIONS: These results obtained in this small and heterogeneous group merit further study in the use of topical 5% imiquimod use in the treatment of intraepithelial neoplasia. An important mechanism of action of imiquimod may involve 2',5'-oligoadenylate synthetase antiviral activity.

Verrucous carcinoma of the foot associated with human papillomavirus type 16.

BACKGROUND: We present 2 patients with verrucous carcinoma (VC) of the foot, a malignancy of unknown origin. OBJECTIVE: Molecular studies from the VCs were undertaken to determine the presence, type, and physical state of human papillomavirus (HPV) as well as the expression levels of certain oncogenes and antioncogenes. METHODS: Synthetic consensus and type-specific primers were used to determine the HPV type from both VCs via polymerase chain reaction (PCR). Verification of fragments was accomplished by means of specific isotope-labeled oligonucleotide probes. The physical state of HPV DNA was determined by two-dimensional gel electrophoresis. Quantitative oncogene and antioncogene expression studies were performed with the use of reverse transcriptase PCR. RESULTS: HPV type 16 was identified in episomal and integrated forms in both tumors. Expression studies revealed increased messenger RNA levels of c-Ki-ras oncogene and the p53 antioncogene and decreased messenger RNA levels of the Rb antioncogene in both VCs. CONCLUSION: Episomal and integrated forms of HPV-16 DNA were found in VCs of the foot, along with alterations of c-Ki-ras, p53, and Rb genes.

Human papillomavirus infections: epidemiology, pathogenesis, and therapy.

BACKGROUND: Human papillomaviruses (HPV) are common human pathogens and are classified into more than 80 different types. These viruses produce benign warts in many cases and aggressive squamous cell carcinomas in other cases. OBJECTIVE: The goal of this review is to update the reader on the epidemiology, pathogenesis, and therapy of HPV infections. Nonanogenital warts are transmitted by skin-to-skin contact while anogenital warts are usually transmitted sexually. Both types of warts produce much morbidity but rarely undergo malignant transformation. They are commonly treated with surgical or cytodestructive therapy, but immunomodulatory agents, such as imiquimod, have been proven to be very effective in anogenital warts and are being evaluated in nonanogenital warts. Other types of HPV have marked oncogenic potential such that over 99% of all cervical cancers and over 50% of other anogenital cancers are due to infection with oncogenic HPV. Many cofactors, such as cigarette smoking, genetics, and helper viruses, have potential roles in HPV oncogenesis, but their relative contributions are poorly understood. Other control measures for warts and HPV-associated cancers are under study, but the greatest future potential may be from the development of prophylactic and therapeutic vaccines. CONCLUSIONS: Infection with HPV is very prevalent as are the clinical manifestations of this family of pathogens. Improved therapies for warts (e.g., imiquimod) have recently become available. Vaccines for HPV offer hope for future interventions for warts as well as for prevention of anogenital malignancies.

The endogenous interferon system in anal squamous epithelial lesions with different grades from HIV-positive individuals.

Anal intraepithelial lesions (ASILs) are considered as precursors of anal cancer. The incidence of high-grade ASIL (HSIL) and progression of low-grade ASIL (LSIL) to HSIL are high in HIV-positive men. Endogenous cytokines, such as interferons (IFNs) play an important role in the regulation of proliferation and immune responses in epithelial cells, and thus, they might control the above-mentioned progression events. Accordingly, we determined mRNA levels of IFN-gamma and IFN-gamma receptors, levels of IFN-gamma receptor-associated kinases (JAK1 and TYK2) and signalling molecules (signal transducer and activator of transcription-1 [STAT1], STAT3, interferon-responsive-factor-1 [IRF-1] and IRF-2) as well as inhibitors of cytokine signalling (protein inhibitor of activated STAT1 [PIAS1] and suppressor of cytokine signalling 2 [SOCS2]) in biopsies of anal condylomas, LSILs as well as HSILs from HIV-positive individuals by a semi-quantitative reverse transcribed polymerase chain reaction (RT-PCR) method. We found that HSIL significantly differs in expression of these genes from LSIL and condylomas. Expression profile of HSIL samples showed activation of STAT3 signalling, probably accounting for the observed high levels of genes that support cellular proliferation (IRF-2, c-fos and c-myc). Decreases in levels of suppressors (IFN-gamma and IRF-1) and JAK1 kinase, but increases in levels of inhibitors of cytokine signalling (PIAS1 and SOCS2) might also contribute to the altered cytokine signalling in HSIL biopsies. These findings might reveal important molecular events associated with progression of LSIL to HSIL in HIV-infected men.

Correlation between mRNA levels of IL-6 and TNF alpha and progression rate in anal squamous epithelial lesions from HIV-positive men.

BACKGROUND: The incidence of anal high-grade intraepithelial lesions (HSILs) and progression of anal low-grade intraepithelial lesions (LSILs) to HSIL are high in HIV-positive men. Endogenous cytokines might support the pathogenesis of this progression. MATERIALS AND METHODS: Accordingly, we determined mRNA levels of IL-6 and TNF alpha and their receptors together with viral genes (HIV-gag and HPV E7) in biopsies of anal condylomas, LSILs and HSILs from HIV-positive individuals by a semiquantitative RT-PCR method. RESULTS: We found that HSIL significantly differs in expression of these genes from LSIL and condylomas, and the latter two lesions were virtually undistinguishable from each other. A correlation between cytokine levels and HIV as well as HPV E7 transcripts suggests that changes might be associated with each other. CONCLUSIONS: These findings reveal important molecular events associated with progression of anal intraepithelial lesions (ASILs) in HIV-infected men.

Human papillomavirus, smoking, and cancer.

BACKGROUND: The effect of smoking on human papillomavirus (HPV) activity and subsequent dysplasia and neoplasia remains controversial. OBJECTIVE: To determine any reported effects of smoking on either HPV activity or HPV-related dysplasia/cancer using retrospective analysis of the literature from 1966 through 1998 via Toxline and PubMed to search for "smoking," "papillomavirus," and "cancer." CONCLUSION: Several recent large studies demonstrated that smoking was associated with a greater incidence of cervical, vulvar, penile, anal, oral, and head and neck cancer in a dose-dependent fashion, while other studies did not show any correlation between smoking and cervical dysplasia after multivariate adjustment. Recent studies have also indicated that smoking may be more closely related to high-grade lesions of the cervix and vulva. These data provide evidence of an association between HPV, smoking, and cancer. Progression of dysplasia likewise seems to be associated with smoking. Several groups have attempted to discern whether the connection between smoking and cervical cancer is from local immunosuppression and/or from direct carcinogenic effects.

Correlation between pretreatment levels of interferon response genes and clinical responses to an immune response modifier (Imiquimod) in genital warts.

Imiquimod (IQ) has been successfully used in treatment of genital warts. In clinical settings, patients responded well but wart reduction rates varied. Our aim was to find a correlation between clinical responses and pretreatment (constitutive) levels of genes that might be involved in the molecular action of IQ. Since IQ is a cytokine inducer, we analyzed levels of expression of genes of the JAK/STAT signaling pathway and their inhibitors as well as interferon response factors (IRFs) in pretreatment biopsy specimens from complete responders (99 to 100% wart reduction rate) versus incomplete responders (75 to 92% wart reduction rate) by reverse transcription-PCR. We found that mRNA levels of signal transducer and activator of transcription 1 (STAT1) and IRF1 were higher in complete responders than in incomplete responders. Incomplete responders expressed larger amounts of STAT3, IRF2, and protein inhibitor of activated STAT1 (PIAS1) mRNAs compared to complete responders before IQ treatment. We hypothesize that high-level expression of STAT1 and IRF1 is advantageous for a better IQ response. The observed differences in constitutive mRNA levels of these genes may be the consequence of alterations in cellular differentiation and/or variable expression of endogenous interferons. Previous in vitro studies showed that keratinocyte differentiation coordinates the balance between positive and negative signals along the JAK/STAT pathway by regulating the IRF1:IRF2 and STAT1:PIAS1 ratios and thus affecting induction of IQ-inducible genes. Specifically, differentiation supports constitutive expression of STAT1 and IRF1 mRNAs but not expression of IRF2 and PIAS1. Our data are in good agreement with studies that showed the importance of STAT1 in cytokine induction and activation of interferon-responsive genes by IQ.

Response of keratinocytes from normal and psoriatic epidermis to interferon-gamma differs in the expression of zinc-alpha(2)-glycoprotein and cathepsin D.

Psoriasis is a T cell-mediated inflammatory disease characterized by hyperproliferation and by aberrant differentiation. We found cathepsin D and zinc-alpha(2)-glycoprotein, two catalytic enzymes associated with apoptosis and desquamation, to be present in the stratum corneum of the normal epidermis but absent from the psoriatic plaque. Psoriasis is characterized by an altered response to interferon-gamma (IFN-gamma), including the induction of apoptosis in normal but not in psoriatic keratinocytes, often with opposite effects on gene expression of suprabasal proteins. We found that IFN-gamma binding and signaling were attenuated in psoriasis: The IFN-gamma receptor, the signal transducer and activator of transcription STAT-1, and the interferon regulatory factor IRF-1 were strongly up-regulated by IFN-gamma in normal keratinocytes, but not in psoriatic ones. IFN-gamma strongly up-regulated the expression of the catalytic enzymes cathepsin D and zinc-alpha(2)-glycoprotein in normal keratinocytes but down-regulated them in psoriatic ones; the reverse was true of the apoptotic suppressor bcl-2. We believe that the aberrant response to IFN-gamma plays a central role in the pathophysiology of psoriasis, particularly the disruption of apoptosis and desquamation.

Enhancement of the innate and cellular immune response in patients with genital warts treated with topical imiquimod cream 5%.

The mechanism of action of imiquimod 5% cream applied topically to patients with genital warts was evaluated in a double-blind, placebo-controlled study. Imiquimod (16 patients) or placebo (three patients) was applied three times per week for up to 16 weeks. All imiquimod-treated patients had a > or =75% reduction in total wart area while only one of three placebo-treated patients had a similar reduction. Wart biopsies were taken at prestudy, week 6, and end of treatment. Polymerase chain reaction (PCR) for human papillomavirus (HPV) DNA and reverse transcriptase (RT)-PCR for messenger (m)RNAs were used to identify cytokines, cellular markers, viral gene products, and cell cycle markers in these biopsies. Treatment with imiquimod, an immune response modifier, stimulated significant increases in mRNA for interferon (IFN)-alpha, IFN-gamma and 2',5' oligoadenylate synthetase (2',5'-AS) as well as a tendency towards increases in tumor necrosis factor (TNF)-alpha and interleukin-12 p40. Significant increases in mRNA for CD4 and a trend toward increases in CD8 were also observed in imiquimod-treated patients, suggesting activation of a cell mediated immune response. Imiquimod administration was also associated with a significant decrease in viral load as measured by HPV DNA and L1 mRNA. The effects on HPV markers were accompanied by an apparent decrease in mRNA expression for markers of cell proliferation and an increase in mRNA for markers of keratinocyte differentiation and tumor suppressors.

Characterization of zinc-alpha(2)-glycoprotein as a cell adhesion molecule that inhibits the proliferation of an oral tumor cell line.

Zn-alpha(2)-glycoprotein (Znalpha(2)gp) is a soluble protein widely distributed in body fluids and glandular epithelia. We have found it to be expressed in stratified epithelia as well. Znalpha(2)gp is clinically correlated with differentiation in various epithelial tumors, including oral and epidermal tumors. We have cloned epidermal Znalpha(2)gp and report the preparation of the recombinant protein in a Baculovirus expression system. Like the native molecule, recombinant Znalpha(2)gp has RNase activity. Znalpha(2)gp functions as a matrix protein for the Tu-138 oral squamous cell carcinoma cell line. Cell attachment to Znalpha(2)gp is comparable to that for fibronectin and is inhibited by the synthetic RGD peptides RGD, RGDV, and RGDS. Attachment is also inhibited by the antibody to integrin alpha(5)beta(1) (the fibronectin receptor), but not by antibodies to integrins alpha(v)beta(3), alpha(3)beta(1), and alpha(2)beta(1). We find that the proliferation of Tu-138 cells is inhibited on a Znalpha(2)gp matrix, as compared with other matrix proteins (fibronectin, vitronectin, laminin, and collagens I and IV) on which growth resembles that on the BSA control. We believe that the role of Znalpha(2)gp in differentiation and its RNase activity are two likely suspects as agents of the inhibition of proliferation.

Zinc-alpha2-glycoprotein expression as a marker of differentiation in human oral tumors.

Zinc-alpha2-glycoprotein (Znalpha2gp) is a soluble major histocompatibility complex homolog widespread in body fluids and in glandular epithelia; the authors recently demonstrated its presence in stratified epithelia. Znalpha2gp has been associated with tumor differentiation in breast cancers and other carcinomas. We compare here its gene expression in histopathologically graded oral squamous cell carcinomas and in their perilesional normals. Znalpha2gp levels are higher in the controls than in the tumors, and higher in well-differentiated tumors than in poorly differentiated ones. Markers of oral epithelial maturation (keratin K13 and involucrin) are less simply related to tumor histology.

Local inflammation may influence oral tumor cell differentiation.

The mRNA levels of keratin K13, involucrin, protein kinase C alpha and epsilon, and interferon-gamma and its receptors were examined in biopsies from human oral squamous cell carcinomas. Expression of all the genes was elevated in the histologically more differentiated tumors, but it was at or below normal (perilesional control) levels in the poorly differentiated ones. For the same set of biopsies, we had previously shown that the well differentiated tumors expressed higher levels of T cell markers. As interferon-gamma stimulates differentiation, its secretion by inflammatory cells at the tumor site may influence the differentiation status of the tumor.

Differentiation-dependent expression of growth regulatory cytokines and their receptors in squamous cell carcinomas of the head and neck.

Cytokines, such as IFN-alpha and TNF-alpha are capable of affecting keratinocyte proliferation in the microenvironment of the tumor. Their elevated expression along with high levels of their receptor mRNAs was determined by a semiquantitative reverse transcription- polymerase chain reaction (RT-PCR) method in biopsies of head and neck squamous cell carcinomas that were established as histologically well or moderately differentiated. In contrast, tumors with poor differentiation exhibited low levels of these growth suppressive factors, although levels of their receptors were elevated. In fact, expression of these growth suppressive cytokines highly correlated with the histological status of tumors suggesting a role of these agents in growth regulation of those tumors. Apparently, growth signaling in these tumors differs in the availability of either the ligand or the receptor.

A guide to immunotherapy of genital warts: focus on interferon and imiquimod.

Genital warts affect at least 1% of sexually active adults. Current therapies are inadequate because they are often painful, may fail to prevent recurrence and transmission of warts, and usually require either surgery or at least application by a physician. Investigation of immunotherapy for genital warts began with interferon. It has been studied in topical, intralesional, systemic and adjuvant applications. We review the major clinical trials of interferon for genital warts, and conclude that intralesional therapy with interferon-alpha or interferon-beta, with complete response rates of 36 to 63%, is the most successful route for interferon monotherapy. In choosing patients for therapy with interferon, major considerations include immune status, pregnancy and ability to return for frequent injections. Imiquimod is a new immune response enhancer that acts through stimulating host cytokine production. Interleukins-1, -2, -6, -8 and -12, interferons alpha, beta and gamma and tumour necrosis factor alpha have all been associated with the mechanism of action of imiquimod. Recently, 3 clinical trials have reported positive results using topical imiquimod to treat genital warts. Complete response rates ranged from 37 to 54% for these controlled trials of 5% imiquimod cream. Adverse effects reported include localised pruritus, erythema, erosion, burning and pain, which were rarely severe enough to cause discontinuation of the medication. Although further trials are necessary to identify the role of imiquimod in the therapy of genital warts, it appears to be an efficacious and well tolerated patient-controlled measure for wart therapy.

Isoforms of cathepsin D and human epidermal differentiation.

Cathepsin D is an ubiquitously expressed lysosomal aspartic proteinase, with well-determined structural and chemical properties but a less clearly defined biological role. In stratified epithelia, the chronology of cathepsin D activation and degradation can be connected with stages of cellular differentiation. We partially purified cathepsin D from human epidermis and from separated stratum corneum by standard biochemical procedures, monitored by SDS-PAGE and Western blotting, and verified its identity as to molecular mass, pH optimum, N-terminal sequencing, reactivity with the specific antibody, inhibition by pepstatin A, and specific enzyme activity. It had hemoglobin-degrading activity over the acid range, with maximum at pH 3. It also degraded bovine serum albumin, human keratins, and stratum corneum extracts at pH 4. We discerned all three isoforms of human cathepsin D (the 52 kDa proenzyme and the active forms at 48 kDa and 33 kDa) in the epidermis; both active forms were also seen in the stratum corneum, but the proenzyme was not. Gene expression of cathepsin D in epidermal keratinocytes resembled that of suprabasal structural proteins (involucrin, keratin K10, transglutaminase) in its response to the calcium switch. An antibody to the 33 kda isoform immunolocalized to the granular layer and the stratum corneum (whereas antibodies to the 48 kDa isoform have been reported to stain mainly the upper spinous and granular layers). A plausible hypothesis to harmonize these results is that cathepsin D is first expressed as the proenzyme in the upper spinous layer, is activated in the lysosomes in the granular layer to the 48 kDa form, and is degraded to the 33 kDa form in the transition zone between the granular layer and the stratum corneum. As the stratum corneum is an acid environment, with an ambient pH of approximately 4.5, cathepsin D is available and suited to contribute to desquamation.

A randomized, controlled, molecular study of condylomata acuminata clearance during treatment with imiquimod.

Imiquimod, an immune response modifier, has been demonstrated to be safe and effective in the treatment of external genital and perianal warts caused by human papillomavirus (HPV). To identify the molecular mechanism(s) by which condylomata acuminata clear during topical treatment with imiquimod, wart skin biopsies were taken from patients before treatment, at treatment week 6, and at the end of treatment. Tissues were analyzed for HPV DNA and for mRNA of several cytokines and HPV gene products. Wart clearance was associated with evidence of tissue production of interferon-alpha, -beta, and -gamma and tumor necrosis factor-alpha. Regression of warts was strongly associated with a decrease in HPV DNA and in mRNA expression for both early and late viral proteins. Thus, topical imiquimod treatment of anogenital warts led to significant increases in local production of multiple interferon mRNAs and a significant reduction in virus load as measured by decreases in HPV DNA and mRNA for early HPV proteins.