H. Randolph Byers

Distinct cellular functions mediated by different VLA integrin α subunit cytoplasmic domains

Abstract To characterize VLA α subunit cytoplasmic domain functions, unaltered α 2 cDNA (called X2C2) and two chimeric cDNAs (called X2C5 and X2C4) were constructed with extracellular α 2 domains and cytoplasmic α 2 , α 5 , and α 4 domains respectively. Upon transfection into rhabdomyosarcoma (RD) cells, each construct yielded comparable expression levels, immunoprecipitation profiles, and avidity for collagen and laminin. However, while RDX2C2 and RDX2C5 transfectants mediated collagen gel contraction, RDX2C4 and a mock transfectant (RDpF) did not. Conversely, only RDX2C4 cells (but not RDX2C2 or RDX2C5) showed enhanced cell migration on collagen and laminin compared with RDpF cells. This indicates markedly differing roles for integrin α subunit cytoplasmic domains in post-ligand binding events. Furthermore, stable exertion of physical force (collagen gel contraction) may involve fundamentally different cellular machinery than the transient adhesion occurring during cell migration. Finally, these findings provide insight into a functional flexibility perhaps resulting from multiple integrins binding to identical ligands.

Organization and Function of Stress Fibers in Cells in Vitro and in Situ

The purpose of this review is to present the tremendous body of research on stress fibers, which has grown exceedingly rapidly in the last 7 or 8 years, due to immunofluorescent techniques, with both a technical and functional perspective. The first section is primarily a chronology of technical innovations which have enabled better observation and characterization of stress fibers. This section also reviews the numerous contractile-associated proteins, actin-binding proteins, regulator proteins, and other proteins shown to localize to stress fibers in a characteristic distribution. The second section discusses the wide variety of roles for stress fibers that have been set forth, including cell spreading, cell adhesion, cell locomotion, contraction, isometric contraction, cell surface compartmentalization, differentiation, cell transformation, tumorigenicity, and morphogenesis. In order to help interpret the significance of the many purported roles of stress fibers, it is important to ask whether stress fibers in vitro are pure artifacts and whether stress fibers exist in cells in situ. This review demonstrates that the stress fiber is fundamentally a light microscopic term, and in order to avoid confusion with other microfilament bundle-containing structures seen in the electron microscope, such as circumferential microfilament bundles, contractile rings, microvilli, microspikes, and rootlet structures, it is necessary to establish criteria for the identification of stress fibers in situ. Finally, using these criteria, this paper presents two recently introduced models which exhibit stress fibers in cells in tissues: the fibroblast, called a scleroblast in the fish scale, and the endothelial cells of avian and mammalian vasculature.

Telomere-based DNA damage responses: a new approach to melanoma

Melanoma is the most fatal skin cancer, often highly resistant to chemotherapy. Here we show that treatment with an 11‐base DNA oligonucleotide homologous to the telomere 3′ overhang sequence (T‐oligo) induces apoptosis of several established human melanoma cell lines, including the aggressive MM‐AN line, whereas normal human melanocytes exposed to the same or higher T‐oligo concentrations show only transient cell cycle arrest, implying that malignant cells are more sensitive to T‐oligo effects. When MM‐AN cells were briefly exposed to T‐oligo in culture and injected into the flank or tail vein of SCID mice, eventual tumor volume and number of metastases were reduced 85‐95% compared with control mice. Similarly, T‐oligos administered intralesionally or sys‐temically selectively inhibited the growth of previously established MM‐AN tumor nodules in the flank and peritoneal cavity by 85 to 90% without detectable toxicity. We previously showed that T‐oligos act through ATM, p95/Nbs1, E2F1, p16INK4A, p53, and the p53 homologue p73 to modulate downstream effectors and now additionally demonstrate striking down‐regulation of the inhibitor of apoptosis protein livin/ML‐IAP. We suggest that T‐oligo mimics a physiologic DNA damage signal that is frequently masked in malignant cells and thereby activates innate cancer prevention responses. T‐oligos may provide a novel therapeutic approach to melanoma.—Puri, N., Eller, M. S., Byers, H. R., Dykstra, S., Kubera, J., Gilchrest, B. A. Te‐lomere‐based DNA damage responses: a new approach to melanoma.

Inhibition of prostate carcinoma establishment and metastatic growth in mice by an antiangiogenin monoclonal antibody.

A neutralizing monoclonal antibody (MAb) 26‐2F to human angiogenin, a potent inducer of neovascularization, has been shown previously to prevent or delay the appearance of angiogenin‐secreting human colon, fibrosarcoma and lung tumor cell xenografts implanted subcutaneously (s.c.) into athymic mice. In an analogous model system, we report here that the antibody also prevents the establishment of PC‐3 androgen‐independent human prostate cancer tumors in, on average, 40% of treated mice (p < 0.0001, survivor analysis). Intriguingly, combining MAb 26‐2F together with cisplatin and suramin, 2 therapeutic agents that together showed little antitumor activity in the aforementioned model, resulted in an even greater degree of protection (71% protected, p = 0.009 compared to antibody treatment alone). This protective effect persisted several weeks after cessation of treatment. Additionally, prophylactic systemic administration of MAb 26‐2F dramatically reduced by 50% the formation of spontaneous regional metastasis originating from primary growth in the prostate gland of PC‐3M cells, highly metastatic variants of PC‐3. Protection from metastasis was still significant when treatment with MAb 26‐2F was delayed until after the primary tumor was well established. The antibody is not directly cytotoxic to either cell type, both of which secrete angiogenin in vitro and when growing as tumors in vivo, but changes the pattern of vascularity in primary tumors growing orthotopically. These findings, together with the observation that angiogenin protein and mRNA are apparently overexpressed in cancerous vs. normal human prostate tissues, demonstrate that angiogenin antagonism represents a promising new approach for preventing progression and metastasis of clinical prostate cancer.

Treatment of small nevomelanocytic nevi with a Q-switched ruby laser

BACKGROUND Small nevomelanocytic nevi are common and some are of cosmetic concern. Conventional therapy may cause a scar or permanent hypopigmentation. OBJECTIVE Our purpose was to determine whether selective photothermolysis of pigmented cells by Q-switched ruby laser treatment removes small nevomelanocytic nevi in a nonscarring fashion. METHODS Twelve patients with 18 small nevomelanocytic nevi were treated with a Q-switched ruby laser (694 nm, 28 nsec) at fluences of 8 J/cm2. Biopsy specimens were obtained after treatment at varying time intervals. RESULTS Twelve lesions (67%) showed a complete response and six lesions (33%) had a partial response. The only adverse sequela observed was mild fibrosis of the papillary dermis, without loss of papillary architecture. CONCLUSION The Q-switched ruby laser is effective in removing small melanocytic nevi. However, some might recur depending on the depth of the nevomelanocytic nests.

Bone morphogenetic protein antagonist noggin promotes skin tumorigenesis via stimulation of the Wnt and Shh signaling pathways

Bone morphogenetic proteins (BMPs) play pivotal roles in the regulation of skin development. To study the role of BMPs in skin tumorigenesis, BMP antagonist noggin was used to generate keratin 14-targeted transgenic mice. In contrast to wild-type mice, transgenic mice developed spontaneous hair follicle-derived tumors, which resemble human trichofolliculoma. Global gene expression profiles revealed that in contrast to anagen hair follicles of wild-type mice, tumors of transgenic mice showed stage-dependent increases in the expression of genes encoding the selected components of Wnt and Shh pathways. Specifically, expression of the Wnt ligands increased at the initiation stage of tumor formation, whereas expression of the Wnt antagonist and tumor suppressor Wnt inhibitory factor-1 decreased, as compared with fully developed tumors. In contrast, expression of the components of Shh pathway increased in fully developed tumors, as compared with the tumor placodes. Consistent with the expression data, pharmacological treatment of transgenic mice with Wnt and Shh antagonists resulted in the stage-dependent inhibition of tumor initiation, and progression, respectively. Furthermore, BMP signaling stimulated Wnt inhibitory factor-1 expression and promoter activity in cultured tumor cells and HaCaT keratinocytes, as well as inhibited Shh expression, as compared with the corresponding controls. Thus, tumor suppressor activity of the BMPs in skin epithelium depends on the local concentrations of noggin and is mediated at least in part via stage-dependent antagonizing of Wnt and Shh signaling pathways.

Modulation of α4β1 and α5β1 integrin expression: Heterogeneous effects of Q-switched Ruby, Nd:YAG, and Alexandrite lasers on melanoma cells in vitro

Integrins of the β1 family are cellular adhesion molecules that play an important role in cell attachment and migration by interacting with extracellular matrix molecules. Agents such as hormones, cytokines, and ultraviolet radiation have all been shown to have an integrin modulating potential. The present study indicates that radiation of Q‐switched lasers is also able to induce transient changes in integrin expression levels on human melanoma cells in vitro.

Expression of PGP 9.5 in granular cell nerve sheath tumors: an immunohistochemical study of six cases.

Background: Protein gene product 9.5 (PGP 9.5) is expressed in brain at 20 to 50 times the levels detected in other organs. Immunohistochemical studies reveal this protein is localized to both central and peripheral neurons. Recently, PGP 9.5 is reported to be a useful marker for cellular neurothekeomas. Herein we test whether PGP 9.5 is a new marker for granular cell nerve sheath tumors.

Pathologic parameters in the diagnosis and prognosis of primary cutaneous melanoma

Significant progress has been made in the last 10 years on the identification of histologic parameters that are independent predictors of melanoma prognosis, immunohistochemical markers of cells of melanocytic origin and changes in adhesion molecules, cytoskeletal proteins, growth factor receptors, cell signaling, and nuclear proliferation proteins associated with tumor progression. Histologic criteria may never be completely sufficient to predict behavior accurately, because the fundamental change that renders a cell aggressive may not be morphologically reflected and may require immunohistochemical or other molecular markers to establish behavior. To date, it is humbling that no immunohistochemical or molecular marker provides a greater predictable value for aggressive behavior than does the simple calibrated ocular micrometer to measure tumor thickness. Nevertheless, development of multiple histologic parameters with the concept of nontumorigenic RGP and tumorigenic VGP provides a reliable statistical model to predict metastases. Fortunately, nontumorigenic RGP melanomas with greater than 75% regression are rare. Thus, individual patients with melanoma without regression and without the tumorigenic VGP can be given reasonable assurance of 100% survival. Nevertheless, this assurance is based on a statistical model with a finite population studied. Additional studies are needed to confirm this model, as well as more definitive markers to precisely predict outcome for those individuals with tumorigenic VGP melanoma.

Attachment, spreading and migration of melanoma cells on vitronectin

Abstract Recent in situ studies suggest the αvβ3 integrin is a tumour progression marker in melanoma. We analyzed 5 human melanoma cell lines for their expression of the vitronectin binding αvβ3 and αvβ5, integrins using flow cytometry. The role of these receptors in cell attachment, spreading and migration was investigated using attachment assays, video time lapse spreading and migration assays and with function blocking monoclonal antibodies. Cell lines derived from later stages of tumor progression exhibited high levels of αvβ3 expression, whereas no similar correlation with αvβ5 expression was identified. Cell attachment, spreading and migration response on vitronectin correlated well with the expression level of the αvβ3 but not the αvβ5 vitronectin receptor. Blocking of the αvβ3 integrin resulted in a significant decrease in cell attachment, spreading and motility whereas the function blocking antibody against the αvβ5 integrin only inhibited cell attachment in cell lines with the highest level of expression of this integrin. Taken together, our study indicates that the level of expression of the αvβ3 and αvβ5 integrins is heterogeneous in melanoma cell lines and that the αvβ5 integrin, if present, may function only during the initial cell attachment whereas the αvβ3 plays an important rôle in cell spreading and cell migration as well.