Karen Hubbard

Mitochondrial deletions in luteinized granulosa cells as a function of age in women undergoing in vitro fertilization

OBJECTIVE To test the hypothesis that mitochondrial DNA (mtDNA) deletions are more prevalent in granulosa cells from women of advanced reproductive age than from younger women undergoing IVF. DESIGN Granulosa cells screened for presence or absence of the 4977-bp deletion in human mtDNA. SETTING University-based fertility clinic. PATIENT(S) Twenty-four women divided equally between two groups: </=34 years old and >/=38 years old. INTERVENTION(S) Patients were given gonadotropin stimulation in preparation for IVF with granulosa cells isolated at the time of follicular aspiration. MAIN OUTCOME MEASURE(S) Presence or absence of the 4977-bp deletion in human mtDNA. RESULT(S) Seven out of 12 women analyzed who were <38 years old and 0 out of 12 women who were >38 years old had normal mtDNA as indicated by the presence of the 4977-bp fragment. CONCLUSION(S) These data suggest that women over the age of 38 have granulosa cells that contain a substantial decrease in the level of normal mitochondria as compared with women </=34 years.

Modulation of the expression of p16INK4a and p14ARF by hnRNP A1 and A2 RNA binding proteins: implications for cellular senescence.

Cellular senescence is a terminal growth phase characteristic of normal human diploid fibroblasts. Altered gene expression during cellular senescence is numerous compared to that of younger proliferative cells in culture. We have previously reported that the levels and activities of hnRNP A1 and A2 RNA binding proteins are decreased in senescent human fibroblasts. Both proteins are multifunctional and may influence the expression of mRNA isoforms during development. In this study, we tested whether overexpression of either protein could modulate the mRNA isoforms of the INK4a locus, specifically p14ARF and p16INK4a. Both INK4a mRNA isoforms have been shown to be growth suppressors and deletions of this locus allow cells to escape cellular senescence. We have found that increasing the ratio of either hnRNP A1 or A2 over that of splicing factor SF2/ASF results in the preferential generation of the p14ARF isoform. Overexpression of A1 or A2 RNA binding proteins also appear to increase the steady state mRNA levels of both isoforms, suggesting that in addition to alternative splicing, A1 and A2 may effect p14ARF and p16INK4a mRNA stability. A constitutive decrease in the ratio of hnRNP A1 or A2 to SF2/ASF in senescent fibroblasts is typically accompanied by an increase in the level of p16INK4a isoform. Our studies suggest that hnRNP A1 and A2 may exert an important role during replicative senescence by altering expression of cell cycle regulatory proteins through mRNA metabolism. J. Cell. Physiol. 193: 19–25, 2002.

Maintenance of telomeres in SV40-transformed pre-immortal and immortal human fibroblasts.

Shortening of telomeres has been hypothesized to contribute to cellular senescence and may play a role in carcinogenesis of human cells. Furthermore, activation of telomerase has frequently been demonstrated in tumor‐derived and in vitro immortalized cells. In this study, we have assessed these phenomena during the life span of simian virus 40 (SV40)‐transformed preimmortal and immortal human fibroblasts. We observed progressive reduction in telomere length in preimmortal transformed cells with extended proliferative capacity, with the most dramatic shortening at late passage. Telomere lengths became stabilized (or increased) in immortal fibroblasts accompanied, in one case, by the activation of telomerase. However, an independent immortal cell line that displayed stable telomeres did not have detectable telomerase activity. Furthermore, we found significant telomerase activity in two preimmortal derivatives. Our results provide further evidence for maintenance of telomeres in immortalized human fibroblasts, but they suggest a lack of causal relationship between telomerase activation and immortalization.

p38 MAP kinase-dependent regulation of the expression level and subcellular distribution of heterogeneous nuclear ribonucleoprotein A1 and its involvement in cellular senescence in normal human fibroblasts.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a RNA binding protein that plays important role in the biogenesis of mRNA, such as alternative splicing and mRNA stability. We have previously demonstrated that hnRNP A1 has diminished protein levels and shows cytoplasmic accumulation in senescent human diploid fibroblasts. Recent reports showed that p38 MAP kinase (p38 MAPK), a member of the MAP kinase family is necessary and sufficient for the cytoplasmic accumulation of hnRNP A1 by stress stimuli such as osmotic shock. p38 MAP kinase has been shown to be involved in cell proliferation and the induction of senescence in response to extracellular stimuli. However, the relationship between hnRNP A1 and p38 MAPK and the roles of hnRNP A1 in cellular senescence have not yet been elucidated. Here we show that hnRNP A1 forms a complex with phospho-p38 MAPK in vivo. Inhibition of p38 MAPK activity with SB203580 elevated hnRNP A1 protein levels and prohibited the cytoplasmic accumulation of the protein, but not hnRNP A2, in senescent cells. The phosphorylation level of hnRNP A1 was elevated in senescent cells. Reduction of hnRNP A1 and A2 levels by siRNA transfection induced a senescence-like morphology and elevated the level of F-actin, a marker of senescence. These results suggest that the expression levels and subcellular distribution of hnRNP A1 are regulated in a p38 MAPK-dependent manner, probably via its phosphorylation. Our results also suggest that hnRNP A2 in addition to hnRNP A1 may play a role in establishing the senescence phenotype.

Patterns of Persistent DNA Damage Associated with Sun Exposure and the Glutathione S-transferase M1 Genotype in Melanoma Patients

Solar radiation can lead to changes affecting DNA metabolism resulting in loss of DNA integrity. Skin specimens obtained from melanoma patients treated at the Memorial Sloan‐Kettering Cancer Center were used to study patterns of DNA fragmentation using the comet assay and levels of deletions in mitochondrial DNA (mtDNA) using real‐time PCR. Skin specimens were classified according to the glutathione S‐transferase M1 (GSTM1) genotype (either wild type [WT] or null) and patient sunburn history. GSTM1 null individuals with a sunburn history showed increased levels of both DNA fragmentation by comet assays and mtDNA deletions relative to GSTM1 WT patients with little or no sunburn history. Microarray analyses identified a number of genes whose expression was upregulated ≥5‐fold in cells from GSTM1‐null patients or from those reporting histories of sunburn. These genes encoded small molecule transporters, various growth factor/chemokine receptors, transcription factors and tumor suppressors. Of 17 genes directly involved in DNA repair, three DNA ligases were highly upregulated while the RAD23 UV excision repair gene and the Growth Arrest and DNA Damage gene (GADD45) were downregulated. These findings support the idea that exposure to solar radiation early in life may induce long‐term cellular changes that lead to persistent DNA damage and altered patterns of gene expression.

Doxorubicin and cyclophosphamide induce cognitive dysfunction and activate the ERK and AKT signaling pathways.

Chemotherapy is associated with long-term cognitive deficits in breast cancer survivors. Studies suggest that these impairments result in the loss of cognitive reserve and/or induce a premature aging of the brain. This study has been aimed to determine the potential underlying mechanisms that induce cognitive impairments by chemotherapeutic agents commonly used in breast cancer. Intact and ovariectomized (OVX) female rats were treated intravenously with either saline or a combination of cyclophosphamide (40 mg/kg) and doxorubicin (4 mg/kg). All subjects were tested for anxiety, locomotor activity, working, visual and spatial memory consecutively. Although anxiety and visual memory were not affected, chemotherapy significantly decreased locomotor activity and impaired working and spatial memory in female rats, independent of their hormonal status. The cognitive deficits observed are hippocampal dependent. Therefore, as a first step to identity the potential signaling pathways involved in this cognitive dysfunction, the protein levels of extracellular signal-regulated kinase 1/2 (Erk1/2), Akt (neuroprotectant) BDNF and (structural protein) PSD95 in hippocampal lysates were measured. Erk1/2 and Akt pathways are known to modulate synaptic plasticity, neuronal survival, aging and cancer. We found an increased activation of Erk1/2 and Akt as well as an increase in the protein levels of PSD95 in OVX female rodents. However, OVX females had a higher overall BDNF level, independent of chemotherapy. These studies provide additional evidence that commonly used chemotherapeutic agents affect cognitive function and impact synaptic plasticity/aging molecules which may be part of the underlying biology explaining cognitive change and can be potential therapeutic targets.

MNK1 Expression Increases During Cellular Senescence and Modulates the Subcellular Localization of hnRNP A1

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA-binding protein that modulates splice site usage, polyadenylation, and cleavage efficiency. This protein has also been implicated in mRNA stability and transport from the nucleus. We have previously demonstrated that hnRNP A1 had diminished protein levels and showed cytoplasmic accumulation in senescent human diploid fibroblasts. Furthermore, we have shown that inhibition of p38 MAPK, a key regulator of cellular senescence, elevated hnRNP A1 protein levels and inhibited hnRNP A1 cytoplasmic localization. In this study, we have explored the possible involvement of MNK1, one of the downstream effector of p38 MAPK, in the regulation of hnRNP A1. We have demonstrated that pharmacological inhibition of MNK1 by CGP 57380 decreased the phosphorylation levels of hnRNP A1 in young and senescent fibroblast cells and blocked the cytoplasmic accumulation of hnRNP A1 in senescent cells. In addition, MNK1 formed a complex with hnRNP A1 in vivo. The expression levels of MNK1, phospho-MNK1, and phospho-eIF4E proteins were found to be elevated in senescent cells. These data suggest that MNK1 regulates the phosphorylation and the subcellular distribution of hnRNP A1 and that MNK1 may play a role in the induction of senescence.

MECHANISM OF IMMORTALIZATION

Model systems implementing various approaches to immortalize cells have led toward further understanding of replicative senescence and carcinogenesis. Human diploid cells have a limited life span, termed replicative senescence. Because cells are terminally growth arrested during replicative senescence, it has been suggested that it acts as a tumor suppression mechanism as tumor cells exhibit an indefinite life span and are immortal. The generation of immortal cells lines, by the introduction of SV40 and human papillomavirus (HPV) sequences into cells, has provided invaluable tools to dissect the mechanisms of immortalization. We have developed matched sets of nonimmortal and immortal SV40 cell lines which have been useful in the identification of novel growth suppressor genes (SEN6) as well as providing a model system for the study of processes such as cellular aging, apoptosis, and telomere stabilization. Thus, their continued use is anticipated to lead to insights into other processes, which are effected by the altered expression of oncogenes and growth suppressors.

Quantification of cell apoptosis for in-vitro colorectal cancer cell cultures based on morphological features

Spatial properties of cell assays obtained by monitoring morphological characteristics of incubated cancer cells are studied as cell apoptosis indicators. Human colon carcinoma cells were cultured in 3d in-vitro microenvironment with FOLFOX treatment to experimentally evaluate cell viability for multiple days. With a 3D cell tracking algorithm guiding an inverted microscope in combination with a digital camera, bright field and fluorescent images of colorectal cells are captured at pre-determined time points. Time course of their spatial randomness properties based on the poisson point process and Voronoi features of cell apoptosis are computed from the fluorescent images. The results show that the heterogeneity among dead cells and the deviation among Voronoi polygon areas for cell apoptoses locations decrease as cell viability decreases. This relationship may be used as a biometric in drug efficacy measurements in in-vitro experiments.

Heterogeneity of colorectal cancer cell positions as a cell viability biometrie

In this paper, we quantify spatial heterogeneity in the positions of live cells as potential biomarkers for cell viability in 3d in-vitro microenvironment under the impact of anti-cancer agents. We present three case studies using human colorectal cancer cell lines of HCT-116, SW-620 and SW-480 in 48-hour experiments. With our data acquisition system guiding an inverted microscope and a digital camera, bright field and fluorescent images are captured at different time points. The viability of the human cancer cells, under FOLFOX impact are measured throughout the experiments. By processing the images captured from microenvironment, spatial heterogeneity in live cell positions are computed based on poisson point process. In all three case studies, we detect an increase in live cell spatial heterogeneity as the cell viability decreases in time as a result of FOLFOX. The encouraging result of correlation between cell viability and spatial heterogeneity has potential to be used in 3D in-vitro microenvironment assays as a biomarker.