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Biomedical subjects

K Butz

Publications and source records attributed to K Butz.

At least 19 recordsLinked to original sources
PubMed2001

p53 mutations are rare events in recurrent cervical cancer.

Mutations of the p53 gene have been shown to be associated with aggressive growth behavior and increased recurrence rates for certain tumors. Primary cervical cancers contain oncogenic human papillomaviruses (HPV) in more than 90% of cases and usually possess wild-type p53 alleles. Cervical cancer cells contain detectable levels of functional p53 protein despite of the expression of the HPV E6 protein, which can induce p53 degradation. Thus, inactivation of p53 by somatic mutation should have functional consequences in HPV-positive cancers. We investigated whether p53 mutations play a role in the recurrence of the disease by analyzing p53 status in 18 biopsy specimens from recurrent cervical cancers. Only one of these (5.6%) contained a p53 mutation, as assessed by a sensitive yeast functional assay that detects mutations of the p53 mRNA between codons 52 and 364. These results indicate that p53 mutations are rare events in recurrent cervical carcinomas, and that somatic mutations of p53 do not provide cervical cancer cells with a selective growth advantage for recurrence.

Adult↗
PubMed2001

Expression pattern of AP-2 transcription factors in cervical cancer cells and analysis of their influence on human papillomavirus oncogene transcription.

The AP-2 family of transcription factors consists of three known members, namely AP-2alpha, AP-2beta, and AP-2gamma. In experimental systems AP-2 factors possess tumor suppressor-like activities, and alterations in the AP-2 expression pattern have been described for some tumor entities. In addition, AP-2 has been implicated in the transcriptional control of human papillomaviruses (HPVs). We investigated here the expression pattern of AP-2alpha, AP-2beta, and AP-2gamma, as well as that of the cellular AP-2 target gene c-erbB-2, in a series of cervical cancer cell lines. In addition, we analyzed the influence of AP-2 factors on the activity of the HPV16 and HPV18 E6/E7 oncogene promoter. We found that, with the exception of HPV-negative C33A cells, all investigated cervical cancer cell lines expressed all three AP-2 family members, although at varying levels. No linear correlation between AP-2 and c-erbB-2 levels was observed. Although AP-2alpha, AP-2beta, and AP-2gamma can activate the c-erbB-2 promoter in reporter gene assays, they do not stimulate the HPV16 or HPV18 E6/E7 promoter. These results indicate that, although a rare event, loss of AP-2 expression occurs in cervical cancer cells. Moreover, AP-2alpha, AP-2beta, and AP-2gamma are neither sufficient nor required to activate the viral E6/E7 promoter.

Antibody Specificity↗
PubMed2001

A validated strategy for side specific prediction of organ confined prostate cancer: a tool to select for nerve sparing radical prostatectomy.

PURPOSE: Nerve sparing radical prostatectomy for prostate cancer should be restricted to patients who harbor tumors without capsular penetration. To our knowledge the selection criteria for nerve sparing radical prostatectomy are not clearly defined. We investigated a panel of preoperative tumor characteristics with respect to their ability to predict organ confined tumor growth for each lobe of the prostate to indicate unilateral or bilateral nerve sparing radical prostatectomy. MATERIALS AND METHODS: Nine preoperative tumor characteristics in 278 patients with clinically localized prostate cancer were included in retrospective univariate and multivariate tree structured regression analysis. The association of clinical stage, serum prostate specific antigen (PSA), PSA density, and results of transrectal ultrasound and systematic sextant biopsy, including a quantitative assessment of cancer in the biopsies with organ confined tumor growth, was statistically evaluated. Except for serum PSA and PSA density preoperative characteristics were considered separately for each prostate lobe. Multivariate analysis results were validated prospectively in 353 patients. RESULTS: On univariate analysis the number of positive biopsies was the most useful single parameter with a positive predictive value of 83% in 274 lobes and a negative predictive value of 55%, followed by mm. of tumor in the biopsy. Of all characteristics included in multivariate analysis only the number of biopsies with high grade cancer, the number of positive biopsies and serum PSA were independent for predicting organ confined cancer. When PSA was less than 10 ng./ml. and not more than 1 biopsy with high grade cancer was identified in a lobe, organ confined tumor growth was present in 86.1% of cases. On prospective validation the same criteria led to an 88.5% incidence of organ confined prostate cancer. Pooling the 2 most favorable groups led to 391 prostate lobes (70.8% of those investigated) with a positive predictive value of 82.1% (95% confidence interval 77.9% to 85.8%). Using the multivariate approach more prostate lobes were assigned to a favorable risk group than on univariate analysis. Clinical stage and simple Gleason grade did not contribute independent information for predicting organ confined disease. CONCLUSIONS: Quantifying cancer and high grade cancer by systematic biopsy and serum PSA concentration are useful preoperative characteristics for predicting organ confined prostate cancer. Side specific analysis of these parameters is a flexible and reliable tool for selecting patients for nerve sparing radical prostatectomy.

Humans↗
PubMed2000

Induction of apoptosis in human papillomaviruspositive cancer cells by peptide aptamers targeting the viral E6 oncoprotein.

Certain types of human papillomaviruses (HPVs) are closely linked to the development of human cancers. Herein, it is shown that intracellular targeting of the HPV16 E6 oncoprotein by E6-binding peptide aptamers resulted in the apoptotic elimination of HPV16-positive cancer cells, whereas HPV-negative cells were not affected. These results provide direct experimental evidence that the HPV E6 oncoprotein has antiapoptotic activity in HPV-positive tumor cells that is required for their survival. The E6-targeting molecules identified herein have implications for the development of therapeutic strategies for the treatment of HPV-associated dysplasias and cancers.

Amino Acid Sequence↗
PubMed2000

Differential cell cycle response of nontumorigenic and tumorigenic human papillomavirus-positive keratinocytes towards transforming growth factor-beta1.

Human papillomaviruses (HPVs) are causative agents of a number of malignancies in humans, including cervical cancer. Their tumorigenic potential is linked to expression of the viral E6/E7 genes which can interfere with normal cell cycle control by targeting p53, p21WAF1, p27KIP1, and pRb. We show here that nontumorigenic and tumorigenic HPV-positive keratinocytes (HPK) exhibit striking differences in the response of cell cycle regulatory genes towards transforming growth factor beta-beta1. Treatment with this agent led to an efficient induction of p53 and the growth-inhibitory p15INK4 and p21WAF1 genes only in nontumorigenic HPKs and was linked to an efficient reduction in viral E6/E7 oncogene expression. This was associated with increased pRb levels, exhibiting sustained hypophosphorylation, and a permanent growth arrest in the G1 phase of the cell cycle. In contrast, tumorigenic HPKs exhibited only a modest rise in p53 protein levels and a substantially reduced induction of the p15INK4 and p21WAF1 genes, which was linked to a lesser degree of viral oncogene repression. In addition, tumorigenic HPKs rapidly resumed cell growth after a transient G1 arrest, concomitantly with the reappearance of hyperphosphorylated pRb. These results support the notion that the progression of HPV-positive cells to a malignant phenotype is associated with increased resistance to growth inhibition by transforming growth factor-beta1. This is linked in the tumorigenic cells to a lack of persistent G1 arrest, inefficient induction of several cell cycle control genes involved in growth inhibition, and inefficient repression of the growth-promoting viral E6/E7 oncogenes.

Blotting, Northern↗
PubMed2000

Peptide aptamers: powerful new tools for molecular medicine.

One of the major goals in molecular medicine is to understand the basis of human diseases at the molecular level and to translate this information into new strategies for diagnosis and therapy. Peptide aptamers represent a novel generation of molecules, which are selected for their intracellular binding to a given target protein. They are useful tools for basic science in blocking the intracellular function of a target protein with high specificity, thereby allowing the study of distinct physiological and pathological processes within living cells. In addition, peptide aptamers provide a basis for the development of novel diagnostic and therapeutic strategies, with implications for a broad variety of different disease entities, including metabolic disorders, infections, and cancer.

Drug Design↗
PubMed1999

Induction of the p53-target gene GADD45 in HPV-positive cancer cells.

The E6 oncoprotein of human papillomaviruses (HPVs) has the potential to functionally antagonize p53. In several experimental model systems, ectopic expression of E6 can block the genotoxic induction of the growth inhibitory p53 target gene gadd45, suggesting that the inactivation of this pathway may play a major role for HPV-associated cell transformation. Here, we investigated whether this reflects the regulation of gadd45 expression in carcinoma-derived HPV-positive cells. We found that the gadd45 gene is efficiently induced by mitomycin C, cisplatin, and UV irradiation in a series of HPV-positive cervical cancer cell lines. Moreover, clear induction of gadd45 gene expression was also observed following treatment with gamma-irradiation, a pathway that is strictly dependent on functional p53. This contrasted with findings in human foreskin keratinocytes experimentally immortalized by expressing the HPV16 E6, E7, or E6/E7 oncogenes from the heterologous CMV promoter, where expression of the E6 gene was linked to a lack of gadd45 induction following gamma-irradiation. These results indicate (1) that the tumorigenic phenotype of HPV-positive cancer cells is not linked to an inability to induce the gadd45 gene following DNA damage, (2) that experimental model systems in which the E6 gene is expressed ectopically and/or in a different cellular context do not necessarily reflect the regulation of p53-associated pathways in HPV-positive cancer cells and (3) that a pathway strictly depending on functional p53 is inducible in HPV-positive cancer cells, providing direct evidence that the endogenous p53 protein in these cells is competent to activate a cellular target gene, despite coexpression of the viral E6 oncogene.

Carcinoma↗
PubMed1998

Uncoupling of p21WAF1/CIP1/SDI1 mRNA and protein expression upon genotoxic stress.

The p21WAF1/CIP1/SDI1 gene is an important regulator of crucial cellular processes, including cell cycle control, cellular differentiation, and the response to genotoxic stress. Induction of p21 gene expression upon DNA damage is widely believed to be p53-dependent. In the present study we analysed the expression of p21 following genotoxic stress, using different DNA-damaging agents and cellular systems. We found that the p21 response markedly varied between different cell lines and also for different genotoxic agents within the same cell line. Genotoxic induction of p21 mRNA expression can occur in the presence of p53-antagonists, such as overexpressed mdm-2 or human papillomavirus (HPV) E6, and in cells harbouring mutated p53 genes. Moreover, upon genotoxic stress, p21 mRNA and protein expression were found to be uncoupled in several cell lines. Thus, transcriptional and postranscriptional changes in p21 expression following DNA damage are not necessarily linked to the intracellular p53 status but strongly depend on the individual cellular background and the type of DNA-damaging agent. Our findings indicate that p21 expression following genotoxic stress underlies a complex control and can be substantially modulated on the posttranscriptional level in a cell-specific manner.

Cells, Cultured↗
PubMed1996

Cellular responses of HPV-positive cancer cells to genotoxic anti-cancer agents: repression of E6/E7-oncogene expression and induction of apoptosis.

The E6 gene of tumor-associated types of human papillomaviruses codes for a functional antagonist of p53. Overexpression of E6 from heterologous promoters can block p53-mediated cellular responses to DNA damage, such as transcriptional stimulation of p53 target genes and cell-cycle arrest in G1. In contrast, genotoxic treatment of HPV-positive cancer cells, which express the E6 gene from chromosomally integrated viral copies, results in increased expression of the p53 target gene p21WAF1 and, in several cell lines, induction of G1 arrest. In the present study, we show that treatment with genotoxic agents, such as mitomycin C and cisplatin, leads to strong repression of viral E6/E7 oncogene expression in HPV16- and HPV18-positive cervical carcinoma cell lines. Kinetic analyses revealed that reduction of E6/E7 expression was not a prerequisite for induction of p21WAF1. We furthermore found that the apoptosis-promoting bax gene could be induced by genotoxic stress in some, but not all, HPV-positive cancer cell lines. Treatment with DNA-damaging agents eventually resulted in apoptotic cell death of HPV-positive cancer cells, irrespective of their capacity to induce the p53 target gene bax. These results support the notion that HPV-positive cancer cells can exhibit intact cellular responses to genotoxic stress, which may involve p53-dependent and -independent biochemical pathways. The ability of HPV-positive cancer cells to induce apoptotic cell death in response to DNA damage could provide a molecular explanation for the therapeutic effects of genotoxic agents in the treatment of cervical cancer.

Apoptosis↗
PubMed1996

Regulation of human papillomavirus transcription by the differentiation-dependent epithelial factor Epoc-1/skn-1a.

Human papillomavirus (HPV) early gene expression is closely linked to the differentiation status of infected epithelial cells. Typically, HPV type 16 (HPV16) or HPV18 E6 and E7 transcripts are only barely detectable within the undifferentiated basal cell layer, but their levels increase concomitantly with higher degrees of epithelial cell differentiation in suprabasal cells. A similar differentiation-dependent distribution of expression has been reported for the recently cloned epithelial cell specific transcription factor Epoc-1/skn-1a. We therefore examined whether Epoc-1/skn-1a may be directly involved in the activation of HPV E6/E7 transcription. Transient transfection studies showed that Epoc-1/skn-1a specifically stimulated the HPV16 and HPV18 E6/E7 promoters. Moreover, ectopically expressed Epoc-1/skn-1a was sufficient to stimulate HPV transcription also in nonepithelial cells. By deletion analyses, the Epoc-1/skn-1a-responsive element was mapped to the promoter-proximal portion of the HPV18 transcriptional control region. Footprint analyses and gel retardation assays demonstrated direct binding of Epoc-1/skn-1a to a hitherto uncharacterized site within this region. Mutation of the Epoc-1/skn-1a recognition site within the context of the complete HPV18 upstream regulatory region inhibited Epoc-1/skn-1a-mediated transactivation. These results show that Epoc-1/skn-1a can directly activate the E6/E7 promoter by binding to the viral transcriptional control region. Thus, Epoc-1/skn-1a may be involved in the differentiation-dependent regulation of HPV transcription.

Base Sequence↗
PubMed1995

Functional p53 protein in human papillomavirus-positive cancer cells.

There is accumulating evidence that the p53 protein contributes to tumor suppression by stimulating the transcription of specific cellular genes, such as the cell cycle control gene WAF1/ClP1. p53-mediated transcriptional activation is inhibited in cotransfection assays by overexpressed E6 protein from cancer-associated human papillomavirus (HPV) types, pointing at a possible molecular mechanism by which these viruses contribute to malignant cell transformation. Here we analysed the transcriptional transactivation function of endogenous p53 protein in a series of cervical cancer cell lines, which express the E6 gene from integrated viral sequences. Transient and stable transfection analyses employing p53-responsive reporter constructs indicated that HPV-positive cervical cancer cells contained transactivating p53 protein. Treatment of HPV-positive cells with genotoxic agents, such as mitomycin C, cisplatin, or u.v. irradiation, resulted in an increase of nuclear p53 protein levels and enhanced binding of p53 to a p53-recognition site. These effects were accompanied by an increase of WAF1/ClP1 mRNA levels. In several HPV-positive cell lines, these molecular events were linked to a cell cycle arrest in G1. In contrast, cancer cells containing mutant p53 genes did not contain transactivating endogenous p53 protein and lacked the p53-mediated response to DNA damaging agents. These results indicate that the tumorigenic phenotype of HPV-positive cancer cell lines does not necessarily correlate with a lack of basal or DNA damage induced p53 activities and that therefore the presence of high risk HPV sequences is not functionally equivalent to the loss of p53 function through somatic mutations of the p53 gene.

Base Sequence↗
PubMed1995

Molecular mechanisms of virus-induced carcinogenesis: the interaction of viral factors with cellular tumor suppressor proteins.

Accumulating evidence indicates that tumor viruses represent a major etiological factor in a significant portion of human cancers. These cancers include human papillomavirus induced anogenital cancers, hepatitis B and C virus associated hepatocellular carcinomas, nasopharyngeal carcinomas and lymphomas linked to Epstein-Barr virus infection, and human T cell leukemia virus associated adult T cell leukemias. This review summarizes the recent progress made in understanding the molecular mechanisms of viral carcinogenesis, with a particular focus on the interaction of viral factors with cellular tumor suppressor proteins. The functional inactivation of tumor suppressor proteins may represent a common strategy by which several tumor viruses contribute to malignant cell transformation.

Cell Cycle↗
PubMed1995

Mutational analysis of adeno-associated virus Rep protein-mediated inhibition of heterologous and homologous promoters.

The four Rep proteins encoded by adeno-associated virus type 2 (AAV-2) inhibit transcription of their own promoters and of several heterologous promoters. To gain insight into the molecular mechanism of Rep-mediated transcription repression, we studied the effects of the four Rep proteins on the accumulation of mRNA transcribed from the human papillomavirus type 18 upstream regulatory region HPV18 URR, the human immunodeficiency virus long terminal repeat, and the AAV-2 p5 and p19 promoters by transient transfection experiments in HeLa cells. We observed a distinct contribution of the C- and N-terminal sequences in which the four Rep proteins (Rep78, Rep68, Rep52, and Rep40) differ from each other. While Rep78 showed a more than 10-fold inhibition of the four promoters studied, transcriptional repression mediated by Rep68 and Rep52 was reduced and nearly completely abolished for Rep40. The contribution of the C terminus of Rep78 was reduced with respect to the inhibition of the AAV-2 p5 and p19 promoters. Point mutations and deletions showed that a C-terminal zinc binding motif is required for zinc binding in vitro but plays no obvious role in the inhibition of homologous and heterologous promoters. Overall, inhibition of the four different promoters was dependent on the identical Rep protein domains with the exception of the AAV-2 p5 promoter. Expression of the AAV-2 p5 promoter was inhibited by a Rep78 protein with a mutation in the nucleotide binding motif, whereas expression of the AAV-2 p19 promoter, the human immunodeficiency virus long terminal repeat, and the HPV18 URR was not. Mutational analysis of the HPV18 URR showed that several, but not a single, cis regulatory elements are involved in the inhibition process. This finding suggests that transcriptional repression is mediated by protein-protein interactions of the Rep proteins either with multiple transcription factors or with target proteins of sequence-specific transcription factors of the basal transcription machinery.

Amino Acid Sequence↗
PubMed1995

[Viruses and cancer: molecular pathologic mechanisms of viral carcinogenesis].

At least 15% of the human cancer incidence is caused by an infection with human tumor viruses. The recent progress of experimental cancer research led to important new concepts about the pathomechanisms of viral carcinogenesis. The functional inactivation of cellular tumor suppressor proteins by viral factors appears to be a key event in the process of virus-associated malignant cell transformation. This review summarizes the current concepts about the interaction between viral oncoproteins and cellular tumor suppressor proteins and evaluates their significance for individual tumor viruses and their associated cancers.

Cell Transformation, Viral↗
PubMed1994

Cellular control of human papillomavirus oncogene transcription.

Specific types of human papillomaviruses (HPVs) are closely associated with the development of cervical cancer. The transforming ability of these high-risk HPV types depends on the expression of the viral E6 and E7 oncogenes. It is therefore of particular interest to elucidate the molecular mechanisms that result in the activation of E6/E7 expression during HPV-associated tumorigenesis. Recently, much progress has been made in characterizing the proteins involved in the regulation of HPV oncogene transcription. This review describes the functional significance of cellular factors involved in the transcriptional control of the E6/E7 promoter for the two most common HPV types associated with cervical cancer, HPV16 and HPV18. In addition, we discuss regulatory pathways that may contribute to the epithelial cells specificity of E6/E7 transcription. The definition of the factors that regulate HPV oncogene transcription could provide new insights into the molecular mechanisms activating viral oncogene expression during cervical carcinogenesis and forms an experimental basis for investigating the specific biochemical pathways that contribute to HPV-associated malignant cell transformation.

Base Sequence↗
PubMed1993

A novel cis-stimulatory element maps to the 5' portion of the human papillomavirus type 18 upstream regulatory region and is functionally dependent on a sequence-aberrant Sp1 binding site.

Gene expression of the cancer-associated human papillomavirus (HPV) type 18 is modulated by cis-regulatory elements located within the viral upstream regulatory region (URR). All cellular factors identified so far involved in viral gene control bind to the 3' portion of the HPV-18 URR. In contrast, very little is known about regulatory elements within the 5' portion of the URR. We therefore analysed this region of unknown function to delineate potential cis-regulatory elements contained therein. By utilizing transient expression assays, an 84 bp fragment could be identified in the 5' portion of the URR that exhibits orientation-independent cis-stimulatory activity in HeLa cervical carcinoma cells and primary human fibroblasts. Gel retardation assays and competition experiments indicated specific binding of cellular proteins to the 84 bp fragment. By functional dissection, a regulatory element with intrinsic cis-stimulatory activity could be mapped within the 84 bp fragment. Binding studies indicate that this cis-stimulatory element contains a sequence-aberrant Sp1 recognition site. Transient luciferase assays performed with mutated templates demonstrate that this Sp1 binding site behaves as a functional Sp1 element in vivo and is a main determinant of the cis-stimulatory activity exerted by the 84 bp fragment. These data show that the 5' portion of the HPV-18 URR contains cis-activating elements and indicate an important functional role for the cellular transcription factor Sp1 in their regulation.

Base Sequence↗
PubMed1993

Transcriptional control of human papillomavirus (HPV) oncogene expression: composition of the HPV type 18 upstream regulatory region.

The malignant transformation potential of high-risk human papillomaviruses (HPVs) is closely linked to the expression of the viral E6 and E7 genes. To elucidate the molecular mechanisms resulting in HPV oncogene expression, a systematic analysis of the cis-regulatory elements within the HPV type 18 (HPV18) upstream regulatory region (URR) which regulate the activity of the E6/E7 promoter was performed. As the functional behavior of a given cis-regulatory element can be strongly influenced by the overall composition of a transcriptional control region, individual elements were inactivated by site-directed mutagenesis in the physiological context of the complete HPV18 URR. Subsequently, the effects of these mutations on the activity of the E6/E7 promoter were assessed by transient transfection assays. We found that the transcriptional stimulation of the E6/E7 promoter largely depends on the integrity of cis-regulatory elements bound by AP1, SP1, and in certain epithelial cells, KRF-1. In contrast to previous reports by implying a key role for NF1 and Oct-1 recognition motifs in the stimulation of papillomavirus oncogene expression, the inactivation of these elements in the context of the HPV18 URR did not strongly affect the transcriptional activity of the E6/E7 promoter. Mutation of a promoter-proximal glucocorticoid response element completely abolished dexamethasone inducibility of the HPV18 E6/E7 promoter and resulted in an increase of its basal activity. Functional dissection of the HPV18 constitutive enhancer region indicates that its transcriptional activity is largely generated by functional synergism between a centrally located AP1 module and thus far undetected cis-active elements present in the 5' flank of the enhancer. Furthermore, comparative analyses using homologous and heterologous promoters show that the transcriptional activity of HPV18 enhancer elements is influenced by the nature of the test promoter in a cell-type-specific manner.

Base Sequence↗