Biomedical subjects

Sandra Caldeira

Publications and source records attributed to Sandra Caldeira.

11 recordsLinked to original sources

PubMed2006

NKIM-6, a new immortalized human brain capillary endothelial cell line with conserved endothelial characteristics.

Primary human brain capillary endothelial cells (hBCECs) are available only in small quantities and have a short life span in vitro; this restricts their use as in vitro model for the blood-brain barrier (BBB). To overcome these limitations, we have established an immortalized hBCEC line (NKIM-6) by transfection with pLXSN16E6E7, which encodes the human papillomavirus type 16 E6 and E7 genes. The cell line exhibits an extended life span in vitro and retains its characteristic endothelial morphology, endothelial markers, and physiology. Likewise, as demonstrated by immunohistochemistry and reverse transcription/polymerase chain reaction (RT-PCR), NKIM-6 cells express BBB markers, and the lack of glial, neuronal, and epithelial markers confirms their endothelial origin. Moreover, with quantitative RT-PCR, we have been able to demonstrate that several ATP-binding cassette-transporters are expressed in NKIM-6 cells with a conserved expression order compared with primary hBCECs. Our results suggest that this cell line might be suitable as in vitro model for several aspects of the BBB.

ATP-Binding Cassette Transporters↗

PubMed2005

Interaction of biomechanical and morphological factors on shoulder workload in industrial paint work.

BACKGROUND: It is known that work-related musculoskeletal disorders are caused by multifactor operations of various risk factors. Among these, the association of these risk factors with pain symptoms and work-related musculoskeletal disorders have been reported in many studies in all typical manufacturing settings (Bernard, 1997). This study focuses on the automobile industry. METHODS: Twenty-nine paint area production workers of an automobile industry plant took part (age 37.7+/-8.2 years; seniority 6.9+/-6.2 years). Musculoskeletal morbidity was assessed through a questionnaire-administered interview and a clinical exam. Each workplace exposure was assessed by the observational rapid upper limb assessment method. The quantification of the workload on shoulders and wrists during the actual work task accomplishment was obtained through surface EMG. FINDINGS: Biomechanical exposure varied significantly between pain symptomatic and asymptomatic workers. It was the only estimator that could predict the risk of occurrence of musculoskeletal symptoms. Multifactor linear analysis showed that some linear and combined anthropometric characteristics could be associated to a higher workload on the shoulders and upper limbs. INTERPRETATION: The rigid external working conditions for employees with different morphologic characteristics, combined with demanding force application associated with the adoption of awkward postures for long and continuous periods of work time, impose constraints in accomplishment of the paint tasks. Additionally our results suggest that the same work task could present different musculoskeletal mechanical load for people with different anthropometrics.

Adult↗

PubMed2005

Skin hyperproliferation and susceptibility to chemical carcinogenesis in transgenic mice expressing E6 and E7 of human papillomavirus type 38.

The oncoproteins E6 and E7 of human papillomavirus type 38 (HPV38) display several transforming activities in vitro, including immortalization of primary human keratinocytes. To evaluate the oncogenic activities of the viral proteins in an in vivo model, we generated transgenic mice expressing HPV38 E6 and E7 under the control of the bovine homologue of the human keratin 10 (K10) promoter. Two distinct lines of HPV38 E6/E7-expressing transgenic mice that express the viral genes at different levels were obtained. In both lines, HPV38 E6 and E7 induced cellular proliferation, hyperplasia, and dysplasia in the epidermis. The rate of occurrence of these events was proportional to the levels of HPV38 E6 and E7 expression in the two transgenic lines. Exposure of the epidermis of nontransgenic mice to UV led to p21WAF1 accumulation and cell cycle arrest. In contrast, keratinocytes from transgenic mice continued to proliferate and were not positive for p21WAF1, indicating that cell cycle checkpoints are altered in keratinocytes expressing the viral genes. Although the HPV38 E6/E7-expressing transgenic mice did not develop spontaneous tumors during their life span, two-stage carcinogen treatment led to a high incidence of papillomas, keratoacanthomas, and squamous-cell carcinomas in HPV38 mice compared with nontransgenic animals. Together, these data show that HPV38 E6 and E7 display transforming properties in vivo, providing further support for the role of HPV38 in carcinogenesis.

Animals↗

PubMed2005

Analysis of E7/Rb associations.

The product of the early gene E7 is one of the major transforming proteins of human papillomaviruses (HPVs). It exerts its activity by associating with and altering the biological functions of several cellular proteins involved in the control of fundamental events, such as cell proliferation and apoptosis. The best-characterized activity of E7 from HPV type 16, the most frequently detected type in cervical cancer, is its ability to bind and induce degradation of the tumor-suppressor retinoblastoma protein (pRb) via the ubiquitin pathway. pRb plays a key role in cell-cycle control by negatively regulating, via direct association, the activity of several transcription factors, including members of the E2F family. The neutralization of pRb functions mediated by E7 results in constitutive activation of the transcription factors, with consequent loss of cell-cycle control. Several studies have shown that the oncogenic potential of a specific HPV type is dependent on the efficiency of E7 in targeting pRb. In this chapter, we describe two methods to measure the efficiency of the E7 proteins from different HPV types in neutralizing the pRb functions. The first one, the plate-binding assay, allows the determination of the pRb binding affinity of E7 proteins, while the second one permits the analysis of their impact on the pRb pathway in intact cells.

3T3 Cells↗

PubMed2004

p53 mutations are common in human papillomavirus type 38-positive non-melanoma skin cancers.

In cervical cells, the E6 protein of the oncogenic human papillomavirus (HPV) types inactivates p53, promoting its degradation. Consequently, mutations of the p53 gene are rarely seen in these cancers. Our recent data indicate that the cutaneous HPV38 is involved in skin carcinogenesis. In this study, we have determined the presence of HPV38 and the status of p53 gene in 32 non-melanoma skin cancers. We found that p53 gene is frequently mutated in HPV38-positive skin cancers and that HPV38 E6 does not promote p53 degradation. Thus, different mechanisms appear to be involved in the development of HPV-positive cervical and skin cancers.

Animals↗

PubMed2004

The short stature homeodomain protein SHOX induces cellular growth arrest and apoptosis and is expressed in human growth plate chondrocytes.

Mutations in the homeobox gene SHOX cause growth retardation and the skeletal abnormalities associated with Léri-Weill, Langer, and Turner syndromes. Little is known about the mechanism underlying these SHOX-related inherited disorders of bone formation. Here we demonstrate that SHOX expression in osteogenic stable cell lines, primary oral fibroblasts, and primary chondrocytes leads to cell cycle arrest and apoptosis. These events are associated with alterations in the expression of several cellular genes, including pRB, p53, and the cyclin kinase inhibitors p21(Cip1) and p27(Kip1). A SHOX mutant, such as seen in Léri-Weill syndrome patients, does not display these activities of the wild type protein. We have also shown that endogenous SHOX is mainly expressed in hypertrophic/apoptotic chondrocytes of the growth plate, strongly suggesting that the protein plays a direct role in regulating the differentiation of these cells. This study provides the first insight into the biological function of SHOX as regulator of cellular proliferation and viability and relates these cellular events to the phenotypic consequences of SHOX deficiency.

Antimetabolites, Antineoplastic↗

PubMed2003

Gene expression patterns in ovarian carcinomas.

We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.

Adenocarcinoma↗

PubMed2003

The role of TP53 in Cervical carcinogenesis.

Functional loss of the tumor suppressor p53 by alterations in its TP53 gene is a frequent event in cancers of different anatomical regions. Cervical cancer is strongly linked to infection by high-risk human papillomavirus (HPV) types. The viral oncoprotein E6 has the ability to associate with and neutralize the function of p53. E6 interacts with a 100-kDa cellular protein, termed E6 associated protein (E6AP; also called ubiquitin-protein ligase E3A or UBE3A), which functions as an ubiquitin protein ligase. The dimeric complex then binds p53 and E6AP catalyzes multi-ubiquitination and degradation of p53. The ability to promote p53 degradation is an exclusive property of E6 from the high-risk HPV types. Indeed, the low-risk E6 proteins lack this activity, although they can bind p53. Consistent with the E6 function of the high-risk HPV types, the majority of cervical cancer cells have a wild-type p53 gene, but the protein levels are strongly decreased. Several independent studies have shown that in a small percentage of cervical tumors the p53 gene is mutated. However, this event appears to be unrelated to the presence or absence of HPV infection and the nature of the tumor.

Female↗

PubMed2003

The E6 and E7 proteins of the cutaneous human papillomavirus type 38 display transforming properties.

Several studies have suggested the involvement of cutaneous human papillomaviruses (HPVs) in the development of nonmelanoma skin cancers. Here we have characterized the in vitro properties of E7 proteins of three cutaneous HPV types, 10, 20, and 38, which are frequently detected in skin specimens. We show that HPV38 E7 is able to inactivate the tumor suppressor pRb and induces loss of G(1)/S transition control, a key event in carcinogenesis. In contrast, HPV10 and HPV20 E7 proteins do not display these in vitro transforming activities. We also show that the two early proteins E6 and E7 of HPV38 are sufficient to corrupt the cell cycle and senescence programs in primary cells, inducing active and long-lasting proliferation of primary human keratinocytes, the natural host cells. Our study shows that E6 and E7 of this cutaneous HPV type have transforming activity in primary human cells, suggesting a role for HPV38 infection in skin carcinogenesis. In further support of such a role, we detected HPV38 DNA in approximately 50% of nonmelanoma skin cancers, but only in 10% of healthy skin specimens (P < 0.001).

3T3 Cells↗

PubMed2002

Human papillomavirus type 32 does not display in vitro transforming properties.

Human papillomavirus type 32 (HPV32) is one of the etiological agents of a benign oral condition, focal epithelial hyperplasia. However, the previously characterized properties of its E7 oncoprotein suggest a possible malignant nature for this virus. In this study we characterized the properties of HPV32 E6 and E7. Our data show that HPV32 E7, despite its high affinity for pRb, does not promote degradation of the cellular protein. In addition, HPV32 E6 does not prevent p53-mediated apoptosis and/or cell cycle arrest. Moreover, coexpression of HPV32 E6 and E7 in primary human fibroblasts or keratinocytes does not alter their proliferative state. Together, these data provide evidence of the benign nature of HPV32.

3T3 Cells↗

PubMed2002

Identification of a novel activity of human papillomavirus type 16 E6 protein in deregulating the G1/S transition.

In this study we show that E6 of human papillomavirus has the ability to deregulate the cell cycle G1/S transition. In rodent immortalized fibroblasts (NIH3T3) serum deprivation or over-expression of the cyclin-dependent kinase inhibitors, p16(INK4a) or p27(KIP1), leads to G1 cell cycle arrest. HPV16 E6 overcomes the antiproliferative signals, gaining the ability to drive serum-deprived and p16(INK4a) or p27(KIP1) over-expressing cells into S phase. E6 protein from the benign HPV type 1 displays a similar activity to HPV16 E6 to deregulate the G1/S transition. Thus, this activity appears to be conserved between E6 proteins from non-oncogenic and oncogenic HPV types. Furthermore, we show that HPV16 E6 is not able to circumvent a G1 arrest imposed by pRb mutant in which all CDK phosphorylation sites have been mutated. These data indicate that the viral protein acts upstream of pRb and its mechanism in promoting cell cycle progression is dependent on pRb phosphorylation. In summary, this study describes a novel biological function of HPV E6 and shows that the S phase entry, required for viral DNA replication, is not exclusively controlled by E7, but that E6 also is involved in this event.

3T3 Cells↗