Mutant p53 gain of function induces HER2 over-expression in cancer cells.

BACKGROUND: HER2 over-expression is related with a poor prognosis in patients with invasive breast cancer tumors. Clinical associations have reported that somatic mutations of p53 more frequently detected in cases of sporadic breast cancer of the HER2 subtypes, besides a high percentage of HER2-amplifying tumors carry germline mutations of p53. The mechanisms responsible for the acquisition of oncogenic functions of p53 mutant proteins (mtp53), known as Gain of Function (GOF), over HER2 expression have not been reported. The objective of this study was to evaluate a possible relationship between p53 mutants and HER2 regulation. METHODS: HER2 expression (transcription and protein), as well as HER2 protein stabilization have been evaluated after inducing or silencing of p53 mutants' expression in cell lines. Finally, we evaluated the interaction of the p53 mutants over the HER2 receptor promoter. RESULTS: Higher HER2 expression in cell lines harboring endogenous mtp53 compared with wt or null expression of p53 cell lines. Transfection of p53 mutants (R248Q and R273C) in cell lines increased the expression of HER2. Silencing of p53 mutants, decrease HER2 expression. The p53 mutants R248Q and R273C significantly increase the luciferase activity on the HER2 promoter, and both mutants also promote acetylation of H3 and H4 histones binding in it. CONCLUSIONS: These findings show for the first time that p53 mutants induce over-expression of HER2 at transcriptional level of the HER2 protein. Our results could have clinical implications in breast cancer and other types of cancer where HER2 is over-expressed and used as a therapy target.

Decreased RARß expression induces abundant inflammation and cervical precancerous lesions.

It is well known that vitamin A and its receptors protect against cancer development and that Retinoid Acid Receptor ß (RARß) is epigenetically silenced during tumoral progression. Cervical Cancer (CC) has been causally linked to high risk human papillomavirus (HR-HPV) infection. However, host factors are important in determining the outcome of persistent HR-HPV infection as most cervical precancerous lesions containing HR-HPVs do not progress to invasive carcinomas. Increasing evidence suggests that low diet in vitamin A and their receptors participate in the development of CC. The aim of this study has been to investigate the effects of abated RARß expression in the development of cervical premalignant lesions in 4 month-old conditional mice (RARß(L-/L-)). Results demonstrated the development of spontaneous squamous metaplasia, inflammatory infiltrate, enhanced mitotic activity, loss of cell differentiation, as well as decreased apoptosis and p16(INK4a) protein levels in RARß(L-/L-) mice cervix. All these changes are hallmarks of moderate dysplasia. Importantly, our results suggest that the low expression of RARß, may induce the down regulation of p16(INK4a), chronic inflammation and decreased apoptosis and may be involved in vulnerability to HR-HPV and early stage cervical carcinogenesis.

P450-aromatase mRNA is expressed in the corpus luteum (CL) of the non-pregnant sheep and goat: the expression of the enzyme is present throughout pregnancy in the goat CL.

In most mammals, the corpus luteum (CL) and placenta are the major sources of progesterone. The goat pregnancy depends on the presence of CL after mid-gestation, while sheep pregnancy does not. The expression and distribution of P450-aromatase (P450-Aro) mRNA throughout gestation has not been investigated in the goat CL and partially in the sheep CL. The present research was designed to characterize the expression of P450-Aro mRNA in small ruminant CL with emphasis in the goat. For this purpose, ovaries from Criollo goats and Pelibuey sheeps were analysed using in situ reverse transcription-polymerase chain reaction (RT-PCR) for the histological detection of P450-Aro transcripts. In addition, P450-Aro expression was determined by in vitro RT-PCR. In situ RT-PCR studies showed that the goat and sheep CL were rich in cells positive for P450-Aro mRNA. We have also found in vitro RT-PCR expression of P450-Aro mRNA in goat CL at 1, 3 and 4 months of gestation. This study shows that the goat CL expresses P450-Aro mRNA along gestation, suggesting that this structure is capable to produce oestrogens up to the end of gestation.

Modulation of apoptosis by early human papillomavirus proteins in cervical cancer.

Cervical cancer (CC) constitutes a major women health problem. Clinical, molecular, and epidemiological investigations have identified persistent infection with high risk human papillomavirus (HR-HPV) as the major cause of CC. HR-HPVs lead to development of cervical carcinoma, predominantly through the action of E5, E6 and E7 viral oncoproteins. After HR-HPV infection, viral proteins employ strategies to modulate apoptosis. The E2 viral protein induces apoptosis in both normal and HPV-transformed cells through activation of caspase-8. The E5 protein can impair CD95L- and TRAIL-mediated apoptosis, which suggests that it may prevent apoptosis at early stages of viral infection. E6 inhibits apoptosis through the proteolytic inactivation of pro-apoptotic proteins such as p53, FADD, or procaspase-8, employing the ubiquitin proteasome pathway, or through interactions with proteins that form the death-inducing signaling complex (DISC) such as TNF-R1. On the other hand, E7 oncoprotein expressing cells are usually predisposed to undergo apoptosis. Useful targets for therapeutic strategies would interfere with expression or function of HR-HPV proteins to eliminate cells that express viral oncoproteins. In this review, we summarize the available data on the interaction of early HPV proteins with cellular factors that promote cell death, and the functional consequences of these interactions on apoptosis.

Impaired cervical homeostasis upon selective ablation of RXRalpha in epithelial cells.

Retinoids play critical regulatory roles in the maintenance of mammalian epithelia and exert pleiotropic effects through nuclear receptors. RXRalpha, which is a ligand-dependent transcription factor, is the most abundant RXR isotype expressed in cervical epithelia, and may play a crucial role in cervix development and homeostasis. We have previously described a mouse model to induce the temporally controlled epithelia-specific somatic mutagenesis of RXRalpha alleles in epidermis. To study the role of RXRalpha in cervical homeostasis, we ablated RXRalpha in cervix epithelial cells of adult mice. We found that such mutant mice develop ectocervical atrophy with moderate epidermoid metaplasia. In addition, we report a simultaneous increase of cell proliferation and apoptosis levels accompanied by alteration in the expression of genes involved in both processes.

Gene expression profile of cervical and skin tissues from human papillomavirus type 16 E6 transgenic mice.

BACKGROUND: Although K14E6 transgenic mice develop spontaneous tumors of the skin epithelium, no spontaneous reproductive tract malignancies arise, unless the transgenic mice were treated chronically with 17beta-estradiol. These findings suggest that E6 performs critical functions in normal adult cervix and skin, highlighting the need to define E6-controlled transcriptional programs in these tissues. METHODS: We evaluated the expression profile of 14,000 genes in skin or cervix from young K14E6 transgenic mice compared with nontransgenic. To identify differentially expressed genes a linear model was implemented using R and the LIMMA package. Two criteria were used to select the set of relevant genes. First a set of genes with a Log-odds > or = 3 were selected. Then, a hierarchical search of genes was based on Log Fold Changes. RESULTS: Microarray analysis identified a total of 676 and 1154 genes that were significantly up and down-regulated, respectively, in skin from K14E6 transgenic mice. On the other hand, in the cervix from K14E6 transgenic mice we found that only 97 and 252 genes were significantly up and down-regulated, respectively. One of the most affected processes in the skin from K14E6 transgenic mice was the cell cycle. We also found that skin from transgenic mice showed down-regulation of pro-apoptotic genes and genes related to the immune response. In the cervix of K14E6 transgenic mice, we could not find affected any gene related to the cell cycle and apoptosis pathways but did observe alterations in the expression of immune response genes. Pathways such as angiogenesis, cell junction and epidermis development, also were altered in their gene expression profiles in both tissues. CONCLUSION: Expression of the HPV16 E6 oncoprotein in our model alters expression of genes that fell into several functional groups providing insights into pathways by which E6 deregulate cell cycle progression, apoptosis, the host resistance to infection and immune function, providing new opportunities for early diagnostic markers and therapeutic drug targets.

Resveratrol decreases Rad51 expression and sensitizes cisplatin­resistant MCF­7 breast cancer cells.

Resveratrol (RES), a polyphenol compound with anti­proliferative properties, has been previously evaluated for its beneficial effects against a variety of tumour cells. The current study elucidated the means by which RES enhances the anti­proliferative effects of cisplatin (CIS) on MCF­7 cells, focusing on the inhibitory effects on DNA repair of double­strand breaks (DSBs). Chemoresistant MCF­7 cells (MCF­7R) were generated by continuous exposure to low concentrations of CIS (10 µM CIS­IC40) during 5 passages, with the IC50 value increasing ~3­fold. Using an MTT assay, we estimated the changes in IC50 for CIS in MCF­7, T47­D, MDA­MB­231 and MCF­7R cells in the presence of RES. The relative transcript level of Nbs­1, Mre­11 and Rad­50 genes was assessed using RT­qPCR analysis. Rad51 and H2AX [pSer139] protein expression was determined by western blot analysis. RES at 50 and 100 µM significantly enhanced the anti­proliferative effects of CIS in both MCF­7 and MCF­7R cells, decreasing the IC50 values for CIS to one­tenth and one­sixth, respectively. A total of 100 µM RES decreased the relative transcript levels of homologous recombination (HR) initiation complex components and the Rad51 protein level in MCF­7 and MCF­7R cells. After 48 h of CIS DNA damage, the levels of Rad51 protein increased, but this effect was inhibited by 100 µM RES. RES also maintained serine 139 phosphorylation of histone H2AX, suggesting that RES prevents the repair of DSBs. It was observed that RES exerts an antagonistic effect over CIS on the activation of Rad51 and sustained phosphorylation of H2AX. The results suggest that RES in combination with DNA damage­based therapy has potential as a strategy to overcome resistance and provide much safer and more effective treatment for breast cancer.

Expression of P450-aromatase in the goat placenta throughout pregnancy.

The enzyme P450-aromatase (P450-Aro) is essential for the conversion of androgens to estrogens. The objective was to study the expression and distribution of P450-Aro in goat placentae throughout pregnancy. For this purpose, we used reverse transcription-polymerase chain reaction (RT-PCR) with primers derived from the ovarian cDNA sequence found by our group. The expression of P450-Aro mRNA was first detected by in vitro RT-PCR in cotyledons at 4 months and was still present at term. Based on in situ RT-PCR, cotyledon microvilli expressed P450-Aro mRNA early in pregnancy; the signal was detected in the syncytiotrophoblast and in non-fused cytotrophoblasts inside the microvilli, but was scarce in the interstitial cells of the villous core. In the last 2 months of pregnancy (including at term), the expression of P450-Aro mRNA was still detected in the syncytiotrophoblast. However, P450-Aro was never detected in the caruncule (regardless of stage of pregnancy). In conclusion, P450-Aro was expressed in the goat placenta microvilli starting early in pregnancy; the expression and distribution of the enzyme increased throughout pregnancy and was still present at term.

[Cervical cancer and DNA microarrays: tumour marker identification]. / Microarreglos de ADN y cáncer cervicouterino: identificación de marcadores tumorales.

Microarray technology has remarkably accelerated the understanding of the molecular events of neoplasias. By means of gene expression profiles, a molecular subclassification of cancer patients and the identification of thousand of genes involved in this pathology have been achieved. Herein, the general use of DNA microarrays in cervical cancer tumorigenesis is reviewed. Finally, putative molecular tumour markers as useful factors in diagnosis, prognosis, and tailor-made therapy for this disease are proposed.

In vivo amplification and rearrangement of c-myc oncogene in human breast tumors.

We have studied the genomic organization of cellular myc (c-myc) proto-oncogene in 48 human primary breast tumors. Two types of alterations (amplification and rearrangement) were observed in 27 (56%) of the tumors studied. The c-myc proto-oncogene appeared to be amplified 2- to 15-fold in the DNA of 20 tumors (41%). Non-germ line c-myc-related fragments (rearrangements) of variable size were detected in 7 primary breast tumors (6 malignant, 1 benign); 4 of these tumors presented both rearrangement and amplification, and the other 3 presented rearrangement only. The majority of the tumors analyzed were invasive ductal adenocarcinomas; 58% of these showed c-myc locus genetic alterations. Although the c-myc alterations described here do not appear to correlate with the aggressive behavior of primary breast tumors, they seem to be associated with development of breast carcinoma.

High correlation between molecular alterations of the c-myc oncogene and carcinoma of the uterine cervix.

We have examined 35 human tumors of the uterine cervix (carcinoma presenting the highest incidence in Mexico; about 34% of women's malignant tumors) for alterations of the cellular myc (c-myc) protooncogene. Elevated amplification and/or rearrangement of the c-myc oncogene were detected in most (approximately 90%) samples (48% showed amplification and 43% presented both alterations). Most tumors were stage II cervical carcinomas and for some of them we detected up to 60-fold amplification of c-myc. These results suggest an important role for c-myc oncogene in the development of tumors of the uterine cervix.

p53 is a rate-limiting factor in the repair of higher-order DNA structure.

The product of the p53 tumor suppressor gene has been implicated in safeguarding genomic stability by transactivating genes involved in cell cycle arrest, repair of DNA damage or induction of apoptosis. Several properties of p53 suggest that it might be directly involved in DNA repair processes. Eukaryotic DNA is highly organized in supercoiled loops anchored to the nuclear matrix. This organization is very important for cell function and survival, suggesting that repair of DNA damage must include both, the integrity of the double helix and the complex DNA topology. In this work, we studied the kinetics and efficiency of higher-order DNA structure repair in cells with normal and reduced levels of p53, and present evidence suggesting that p53 may be involved in the stabilization and/or repair of higher-order DNA structure.

Isolation and partial characterization of 2-microns yeast plasmid as a transcriptionally active minichromosome.

Yeast cell extracts from 2-microns-containing strains (cir+) showed higher transcriptional activity than their corresponding isogenic sets (cir0). These extracts were used to purify transcriptionally active 2-microns minichromosomes in a sucrose gradient. Minichromosomes were transcribed in vitro and, employing hybridization techniques, the RNA synthesized was shown to present 2-microns-specific sequences. This model system should permit the direct study of transcriptionally active eucaryotic chromatin.

Human papilloma virus status and chromosomal imbalances in primary cervical carcinomas and tumour cell lines.

Human papilloma virus (HPV) infection is the crucial step in the initiation of cervical carcinomas. In addition, HPV18 has been implicated in tumour progression and adverse clinical outcome. We determined the HPV types in 12 primary cervical carcinomas and 12 cell lines and compared the findings with the comparative genetic hybridisation (CGH) pattern of chromosomal alterations. The most frequent alteration was the deletion at 3p14 followed by the loss of 2q34-q36 along with 3q gain. High risk HPV types were detected in all samples except one primary tumour. In contrast to the normal distribution, HPV18 was present in 75% of cases including all cell lines. The cell lines carried a higher number of genetic alterations and a different CGH pattern for several chromosomes than the primary tumours, despite microdissection. Purely HPV18 positive cases indicated a high incidence of imbalances at specific loci with peaks of the histogram coinciding with known HPV integration sites. The study suggests that HPV infection is associated with a recurrent pattern of chromosomal changes in cervical carcinomas and that the development and progression of these alterations is triggered by integration into the host genome.

Transcriptional repression in normal human keratinocytes by wild-type and mutant p53.

Wild-type p53 is a nuclear phosphoprotein that inhibits cell proliferation and represses transcriptionally most TATA box-containing promoters in transformed or tumor-derived cell lines. This study demonstrates that p53 alters transcription of the long control region (LCR) of human papillomavirus type 18 (HPV-18). Wild-type and mutant p53 143Val to Ala repressed the HPV-18 LCR promoter in normal human keratinocytes, the natural host cell for HPV infections. Repression by wild-type p53 was also observed in C-33A cells and in an HPV-16-immortalized cell line with an inducible wild-type p53. However, when C-33A cells were cotransfected with the HPV-18 LCR and mutant 143Val to Ala, repression did not occur. Mutant p53 135Cys to Ser did not induce repression in either normal human keratinocytes or in the C-33A line; although like 143Val to Ala, it is thought to affect the DNA binding activity of the wild-type protein. The ability of mutant p53 143Val to Ala to inactivate the HPV early promoter in normal cells (by approximately 60% reduction) suggests that this mutant may be able to associate with wild-type p53 and interact with TATA box-binding proteins. Therefore, these results demonstrate that the transcriptional activities of p53 mutants may be dependent upon the cell type assayed and the form of its endogenous p53. Furthermore, normal human keratinocytes represent an alternative model for determining the activities of p53 mutants.

Novel combination of c-myc, N-myc and N-ras oncogene alteration in brain tumors.

We have examined forty human brain tumors (neoplasias presenting an important incidence in Mexico), for cellular myc (c-myc), N-myc and N-ras proto-oncogene alterations. An elevated amplification and/or rearrangement of the oncogenes was detected in most samples (60% presenting alteration for c-myc, 54% for N-myc, 6% for N-ras and 60% for ras-related genes). The tumors were of different histological types and for some of them we detected either amplification and/or rearrangement of the oncogenes. We describe, for the first time, the alterations of two related genes (c-myc and N-myc) in the same tumor samples; in 64% of the analyzed samples, oncogene alterations were accompanied by enhanced expression of N-myc and ras-related genes. These results suggest an important role for c-myc, N-myc and N-ras oncogenes, in the development and progression of brain tumors.

A closer look at specific therapeutic strategies in leukemia.

Leukemia-associated fusion genes are detected in a significant proportion of newly diagnosed cases, where genes encoding transcription factors are usually found at one of the breakpoints. Activated fusion proteins, such as PML-RARalpha and AML1-ETO, have been shown to inhibit cellular differentiation by recruitment of nuclear corepressor complexes, which maintain local histone deacetylase (HDAC) in a variety of hematologic lineage-specific gene promoters. This HDAC-dependent transcriptional repression appears as a common pathway in the development of leukemia and could represent an important target for new therapeutic agents. On the other hand, the Bcr-Abl oncoprotein shows high tyrosine kinase activity and deregulates signal transduction pathways involved normally in both apoptosis and proliferation. This aberrant activity is affected by signal transduction inhibitors (STIs), which block or prevent the oncogenic pathway. In this review, we present a closer look at our understanding of both the reversible transcriptional repression controlled by HDAC and the deregulated Bcr-Abl signal transduction. In addition, the application of low molecular weight drugs for human leukemia treatment based in this knowledge results in durable clinical remission and acceptable risk of toxic effects that should increase the cure rate. We hope that this review will provide timely information to the readers.

The retinoblastoma gene product negatively regulates cellular or viral oncogene promoters in vivo.

The protein product of the retinoblastoma tumor suppressor gene (pRb) has been demonstrated to bind transcriptional factors such as E2F and c-Myc protein in vitro. To determine whether pRb regulates both cellular (c-myc) and viral (HPV LCR) promoter activity in vivo, MYC-CAT or HPV LCR-CAT chimeric expression plasmids were generated and cotransfected with a pCMV-RB expression plasmid. pRb repressed both myc and LCR transcription but not SV40-CAT. Transcriptional repression induced by pCMV-RB was relieved by addition of pSV2-E7. Moreover, immunohistochemical assays indicate that in cervical intraepithelial neoplasia lesions, an increased pRb expression correlates with decreased c-myc oncogene expression. These results suggest that pRb can negatively regulate c-myc transcription in vivo in both normal tissue or early cervical lesions. However, in HPV induced invasive cervical carcinomas where viral DNA is integrated expressing its oncoproteins, pRb could be complexed by HPV E7 oncoprotein releasing the repression effect and promoting cell growth.

Therapeutic uterine-cervix cancer vaccines in humans.

Infection with high-risk human papillomavirus (HPV) types is involved in early stages of uterine=cervix cancer development. The virally encoded E6 and E7 oncoproteins behave as tumor-specific antigens and represent targets for a vaccine designed to control HPV-induced tumors. Using either proteins or peptides based on E6 and E7 oncoproteins of HPV16 and 18, phase I clinical trials of therapeutic vaccines against HPV-associated cervical cancers have recently been reported. Although the effectiveness of these vaccines cannot be evaluated in such small studies, they constitute an important step toward the development of therapeutic uterine=cervix cancer vaccines. A polytope DNA vaccination approach combined with immunomodulatory cytokines may offer an excellent strategy to reduce the risk of relapse and metastasis following conventional therapies.

Different arrangement of human papillomavirus E2 binding sites distinguishes cutaneous types from those associated with mucosal lesions.

High-risk-type human papillomavirus DNA sequences are found in a high percentage of carcinomas from the uterine-cervix, with the viral E1-E2 gene region usually disrupted and the E6 and E7 oncoproteins consistently expressed. The E2 protein is known to repress early transcription from genital HPV promoters having a proximal E2 binding site (E2BS) close to the TATA box. On the contrary, the E2 protein activates cutaneous early promoters having a longer distance between these sites. Using an in vivo approach we analyzed the regulation, by the BPV-1E2 protein, of a natural HPV-18 promoter where proximal E2BS were placed at variable positions relative to the TATA box, and of heterologous promoters where E2BS was placed upstream of any other known DNA-binding elements. Our results confirm that the E2 protein represses or activates HPV early gene transcription depending on the distance between the TATA box and the proximal E2BS.