David S. Cassarino

Module Map of Stem Cell Genes Guides Creation of Epithelial Cancer Stem Cells

Self-renewal is a hallmark of stem cells and cancer, but existence of a shared stemness program remains controversial. Here, we construct a gene module map to systematically relate transcriptional programs in embryonic stem cells (ESCs), adult tissue stem cells, and human cancers. This map reveals two predominant gene modules that distinguish ESCs and adult tissue stem cells. The ESC-like transcriptional program is activated in diverse human epithelial cancers and strongly predicts metastasis and death. c-Myc, but not other oncogenes, is sufficient to reactivate the ESC-like program in normal and cancer cells. In primary human keratinocytes transformed by Ras and I kappa B alpha, c-Myc increases the fraction of tumor-initiating cells by 150-fold, enabling tumor formation and serial propagation with as few as 500 cells. c-Myc-enhanced tumor initiation is cell-autonomous and independent of genomic instability. Thus, activation of an ESC-like transcriptional program in differentiated adult cells may induce pathologic self-renewal characteristic of cancer stem cells.

An evaluation of the role of mitochondria in neurodegenerative diseases : Mitochondrial mutations and oxidative pathology, protective nuclear responses, and cell death in neurodegeneration

There is mounting evidence for mitochondrial involvement in neurodegenerative diseases including Alzheimers and Parkinsons disease and amyotrophic lateral sclerosis. Mitochondrial DNA mutations, whether inherited or acquired, lead to impaired electron transport chain (ETC) functioning. Impaired electron transport, in turn, leads to decreased ATP production, formation of damaging free-radicals, and altered calcium handling. These toxic consequences of ETC dysfunction lead to further mitochondrial damage including oxidation of mitochondrial DNA, proteins, and lipids, and opening of the mitochondrial permeability transition pore, an event linked to cell death in numerous model systems. Although protective nuclear responses such as antioxidant enzymes and bcl-2 may be induced to combat these pathological changes, such a vicious cycle of increasing oxidative damage may insidiously damage neurons over a period of years, eventually leading to neuronal cell death. This hypothesis, a synthesis of the mitochondrial mutations and oxidative stress hypotheses of neurodegeneration, is readily tested experimentally, and clearly points out many potential therapeutic targets for preventing or ameliorating these diseases.

The parkinsonian neurotoxin MPP+ opens the mitochondrial permeability transition pore and releases cytochrome c in isolated mitochondria via an oxidative mechanism

The mitochondrial transition pore (MTP) is implicated as a mediator of cell injury and death in many situations. The MTP opens in response to stimuli including reactive oxygen species and inhibition of the electron transport chain. Sporadic Parkinsons disease (PD) is characterized by oxidative stress and specifically involves a defect in complex I of the electron transport chain. To explore the possible involvement of the MTP in PD models, we tested the effects of the complex I inhibitor and apoptosis-inducing toxin N-methyl-4-phenylpyridinium (MPP+) on cyclosporin A (CsA)-sensitive mitochondrial swelling and release of cytochrome c. In the presence of Ca2+ and Pi, MPP+ induced a permeability transition in both liver and brain mitochondria. MPP+ also caused release of cytochrome c from liver mitochondria. Rotenone, a classic non-competitive complex I inhibitor, completely inhibited MPP(+)-induced swelling and release of cytochrome c. The MPP(+)-induced permeability transition was synergistic with nitric oxide and the adenine nucleotide translocator inhibitor atractyloside, and additive with phenyl arsine oxide cross-linking of dithiol residues. MPP(+)-induced pore opening and cytochrome c release were blocked by CsA, the Ca2+ uniporter inhibitor ruthenium red, the hydrophobic disulfide reagent N-ethylmaleimide, butacaine, and the free radical scavenging enzymes catalase and superoxide dismutase. MPP+ neurotoxicity may derive from not only its inhibition of complex I and consequent ATP depletion, but also from its ability to open the MTP and to release mitochondrial factors including Ca2+ and cytochrome c known to be involved in apoptosis.

Cybrids in Alzheimer's disease: a cellular model of the disease?

The mitochondrial electron transport chain enzyme cytochrome c oxidase (COX) is defective in patients with sporadic Alzheimers disease(AD). This defect arises from the mutation of mitochondrial DNA (mtDNA). To develop a tissue culture system that would express this genetically derived bioenergetic lesion and permit characterization of its functional consequences, we depleted Ntera2/D1 (NT2) teratocarcinoma cells of endogenous mtDNA and repopulated them with platelet mtDNA from AD patients. Cytochrome c oxidase activity was depressed in the resulting AD cytoplasmic hybrids (cybrids) compared with cybrids prepared with mtDNA from non-AD controls. Reactive oxygen species (ROS) production and free radical scavenging enzyme activities were significantly elevated in AD cybrids. A COX defect in NT2 AD cybrid lines indicates that AD patients possess mtDNA COX gene mutations that are sufficient for determining this biochemical lesion. Expression of unique functional characteristics (increased ROS production and free radical scavenging enzyme activities) relevant to neurodegeneration demonstrates the utility of these cells in defining AD pathophysiology at a cellular level. This in vitro tissue culture model of AD may prove useful in drug screening.

Cutaneous squamous cell carcinoma: a comprehensive clinicopathologic classification. Part One

Cutaneous squamous cell carcinoma (SCC) includes many subtypes with widely varying clinical behaviors, ranging from indolent to aggressive tumors with significant metastatic potential. However, the tendency for pathologists and clinicians alike is to refer to all squamoid neoplasms as generic SCC. No definitive, comprehensive clinicopathological system dividing cutaneous SCCs into categories based upon their aggressiveness has yet been promulgated. Therefore, we have proposed the following based upon the malignant potential of SCC variants, separating them into categories of low (≤2% metastatic rate), intermediate (3–10%), high (greater than 10%), and indeterminate behavior. Low‐risk SCCs include SCC arising in actinic keratosis, HPV‐associated SCC, tricholemmal carcinoma, and spindle cell SCC (unassociated with radiation). Intermediate‐risk SCCs include adenoid (acantholytic) SCC, intraepidermal epithelioma with invasion, and lymphoepithelioma‐like carcinoma of the skin. High‐risk subtypes include de novo SCC, SCC arising in association with predisposing factors (radiation, burn scars, and immunosuppression), invasive Bowens disease, adenosquamous carcinoma, and malignant proliferating pilar tumors. The indeterminate category includes signet ring cell SCC, follicular SCC, papillary SCC, SCC arising in adnexal cysts, squamoid eccrine ductal carcinoma, and clear‐cell SCC. Subclassification of SCC into these risk‐based categories, along with enumeration of other factors including tumor size, differentiation, depth of invasion, and perineural invasion will provide prognostically relevant information and facilitate the most optimal treatment for patients.

Pramipexole reduces reactive oxygen species production in vivo and in vitro and inhibits the mitochondrial permeability transition produced by the parkinsonian neurotoxin methylpyridinium ion.

Abstract: Sporadic Parkinsons disease is associated with a defect in the activity of complex I of the mitochondrial electron transport chain. This electron transport chain defect is transmitted through mitochondrial DNA, and when expressed in host cells leads to increased oxygen free radical production, increased antioxidant enzyme activities, and increased susceptibility to programmed cell death. Pramipexole, a chemically novel dopamine agonist used for the treatment of Parkinsons disease symptoms, possesses antioxidant activity and is neuroprotective toward substantia nigral dopamine neurons in hypoxic‐ischemic and methamphetamine models. We found that pramipexole reduced the levels of oxygen radicals produced by methylpyridinium ion (MPP+) both when incubated with SH‐SY5Y cells and when perfused into rat striatum. Pramipexole also exhibited a concentration‐dependent inhibition of opening of the mitochondrial transition pore induced by calcium and phosphate or MPP+. These results suggest that pramipexole may be neuroprotective in Parkinsons disease by attenuating intracellular processes such as oxygen radical generation and the mitochondrial transition pore opening, which are associated with programmed cell death.

Mitochondria in Sporadic Amyotrophic Lateral Sclerosis

Mitochondria are abnormal in persons with amyotrophic lateral sclerosis (ALS) for unknown reasons. We explored whether aberration of mitochondrial DNA (mtDNA) could play a role in this by transferring mitochondrial DNA (mtDNA) from ALS subjects to mtDNA-depleted human neuroblastoma cells. Resulting ALS cytoplasmic hybrids (cybrids) exhibited abnormal electron transport chain functioning, increases in free radical scavenging enzyme activities, perturbed calcium homeostasis, and altered mitochondrial ultrastructure. Recapitulation of defects previously observed in ALS subjects and ALS transgenic mice by expression of ALS mtDNA support a pathophysiologic role for mtDNA mutation in some persons with this disease.

Interaction Among Mitochondria, Mitogen‐Activated Protein Kinases, and Nuclear Factor‐κB in Cellular Models of Parkinson's Disease

Abstract: Oxidative stress induced by acute complex I inhibition with 1‐methyl‐4‐phenylpyridinium ion activated biphasically the stress‐activated c‐Jun N‐terminal kinase (JNK) and the early transcription factor nuclear factor‐κB (NF‐κB) in SH‐SY5Y neuroblastoma cells. Early JNK activation was dependent on mitochondrial adenine nucleotide translocator (ANT) activity, whereas late‐phase JNK activation and the cleavage of signaling proteins Raf‐1 and mitogen‐activated protein kinase (MAPK) kinase (MEK) kinase (MEKK)‐1 appeared to be ANT‐independent. Early NF‐κB activation depended on MEK, later activation required an intact electron transport chain (ETC), and Parkinsons disease (PD) cybrid (mitochondrial transgenic cytoplasmic hybrid) cells had increased basal NF‐κB activation. Mitochondria appear capable of signaling ETC impairment through MAPK modules and inducing protective NF‐κB responses, which are increased by PD mitochondrial genes amplified in cybrid cells. Irreversible commitment to apoptosis in this cell model may derive from loss of Raf‐1 and cleavage/activation of MEKK‐1, processes reported in other models to be caspase‐mediated. Therapeutic strategies that reduce mitochondrial activation of proapoptotic MAPK modules, i.e., JNK, and enhance survival pathways, i.e., NF‐κB, may offer neuroprotection in this debilitating disease.

Inhibition by R(+) or S(–) pramipexole of caspase activation and cell death induced by methylpyridinium ion or beta amyloid peptide in SH‐SY5Y neuroblastoma

Cell models of neurodegenerative diseases (NDD) can involve expression of mutant nuclear genes associated with Mendelian forms of the diseases or effects of toxins believed to replicate essential disease features. Death produced by exposing neural cells to methylpyridinium ion (MPP+) or neurotoxic beta amyloid (BA) peptides is frequently used to study features of the sporadic, most prevalent forms of Parkinsons disease (PD) and Alzheimers disease (AD), respectively. We examined in replicating SH‐SY5Y human neuroblastoma cells the release of cytochrome C into cytoplasm, activation of caspases 9 and 3, and loss of calcein retention as markers of the “mitochondrial” pathway of cell death. Exposure to 5 mM MPP+, which induces apoptotic cell death within 18–24 hr, released cytochrome C within 4 hr, activated caspases 9 and 3, and reduced calcein accumulation. BA 25–35 peptide produced more rapid and greater elevations of caspase 3 activity; no effects were observed with the nontoxic BA 35–25 reverse sequence. The dependence on mitochondrial transition pore (MTP) activity of MPP+‐induced caspase activations was demonstrated by preincubation with bongkreckic acid, which blocked elevations of caspases 9 and 3. Stereoisomers of pramipexole (PPX), a free radical scavenger and inhibitor of MTP opening, inhibited caspase activation (MPP+ and BA) and restored calcein accumulation (MPP+). Our results demonstrate that MPP+ and BA can induce cell death through MTP‐dependent activation of caspase cascades. PPX stereoisomers interfere with activation of these cell death pathways and may be useful clinically as neuroprotectants in PD and AD and related diseases.

Mitochondrial dysfunction in cybrid lines expressing mitochondrial genes from patients with progressive supranuclear palsy.

Abstract: Progressive supranuclear palsy (PSP) is a neurodegenerative movement disorder of unknown etiology. We hypothesized that mitochondrial DNA (mtDNA) aberration could occur in this disease and contribute to its pathogenesis. To address this we created transmitochondrial cytoplasmic hybrid (cybrid) cell lines expressing mitochondrial genes from persons with PSP. The presence of cybrid mtDNA aberration was screened for by biochemical assay of mitochondrial gene products. Relative to a control cybrid set, complex I activity was reduced in PSP cybrid lines (p < 0.005). Antioxidant enzyme activities were elevated in PSP cybrid lines. These data suggest that mtDNA aberration occurs in PSP, causes electron transport chain pathology, and can produce oxidative stress. Further study of mitochondrial dysfunction in PSP may yield insights into why neurodegeneration occurs in this disease.