Clotrimazole inhibits cell proliferation in vitro and in vivo.

Cell proliferation is critically dependent on the regulated movement of ions across various cellular compartments. The antimycotic drug clotrimazole (CLT) has been shown to inhibit movement of Ca2+ and K+ across the plasma membrane. Our results show that CLT inhibits the rate of cell proliferation of normal and cancer cell lines in a reversible and dose-dependent manner in vitro. Moreover, CLT depletes the intracellular Ca2+ stores and prevents the rise in cytosolic Ca2+ that normally follows mitogenic stimulation. In mice with severe combined immunodeficiency disease (SCID) and inoculated intravenously with MM-RU human melanoma cells, daily subcutaneous injections of CLT induced a significant reduction in the number of lung metastases. Modulation of early ionic mitogenic signals and potent inhibition of cell proliferation both in vitro and in vivo are new and potentially useful clinical effects of CLT.

Stress fibers in cells in situ: immunofluorescence visualization with antiactin, antimyosin, and anti-alpha-actinin.

Stress fiber-like patterns are visualized by indirect immunofluorescence in scleroblasts (fibroblasts) in situ on the scale of the common goldfish, Carassius auratus, using an affinity-purified antiactin, antimyosin, and anti-alpha-actinin. These fibers demonstrate the classical convergent and parallel patterns exhibited by stress fibers in tissue culture cells. Because the dimensions, the composition, and the pattern of distribution of these cytoplasmic fibers correspond well with those of stress fibers in cultured cells, we will call these fibers stress fibers also. The staining patterns with anti-alpha-actinin and antimyosin along the stress fibers often reveal a periodicity of 1-2 microM, identical to that found in cells in vitro. The majority of scleroblasts do not exhibit stress fiber staining and they are specifically located in the central regions of the scale. Stress fibers are present in scleroblasts residing on or near the edges or radical ridges of the scale. They are consistently orientated perpendicular to these structures; however, unlike microtubules, stress fibers show no co-alignment with collagen fibers of the scale. The finding that stress fibers are located in regions of the scale more subject to shearing forces may indicate their role in increased cellular adhesion to the substratum.

Transformations in the structure of the cytoplasmic ground substance in erythrophores during pigment aggregation and dispersion. I. A study using whole-cell preparations in stereo high voltage electron microscopy.

Pigment migration in cultured erythrophores of the squirrel fish Holocentrus ascensionis, after manipulation with K+, epinephrine, 3',5'-dibutyryl cyclic adenosine monophosphate, theophylline, and caffeine, is essentially identical to that observed in this chromatophore in situ. For such observations, the erythrophores are dissociated from the scales with hyaluronidase and collagenase, and allowed to spread on an amorphous collagen substrate, where they resemble the discoid erythrophore in situ. In this state, they are readily fixed by glutaraldehyde and osmium tetroxide, and are then critical-point dried for whole-cell viewing in the high voltage electron microscope. The organization and fine structure of the erythrophore cytoplast was stereoscopically examined after fixation of the pigment granules in four experimental states: pigment dispersed, pigment aggregated, pigment aggregating, and pigment dispersing. In the dispersed cell, granules are contained in an extensive three-dimensional lattice composed of radially oriented microtubules and a network of fine filaments 3-6 nm in diameter (microtrabeculae), whereas in the aggregated cell, the microtrabecular system is absent, and the majority of the microtubules appear displaced into the cortices on the cytoplasmic surface of the plasma membrane. In cells fixed while aggregating, few microtrabeculae are observed, although formless thickenings are observed in the cortices, on granules, and between clumped granules. In dispersing cells, the microtrabecular system is reformed from material stored in the cortices and with the granules in the centrosphere. These observations suggest that the granules are suspended in a dynamic microtrabecular system that withdraws during pigment aggregation and is restructured during pigment dispersion. The microtubules guide linear granule motion not by defining physical channels, but by a recognizable affinity of microtubules, microtrabeculae, and granules for one another.

CD44H regulates tumor cell migration on hyaluronate-coated substrate.

CD44 is a broadly distributed cell surface glycoprotein expressed in different isoforms in various tissues and cell lines. One of two recently characterized human isoforms, CD44H, is a cell surface receptor for hyaluronate, suggesting a role in the regulation of cell-cell and cell-substrate interactions as well as of cell migration. While CD44H has been shown to mediate cell adhesion, direct demonstration that CD44H expression promotes cell motility has been lacking. In this work we show that a human melanoma cell line, stably transfected with CD44H, displays enhanced motility on hyaluronate-coated surfaces while transfectants expressing an isoform that does not bind hyaluronate, CD44E, fail to do so. Migration of CD44H-expressing transfectants is observed to be blocked by a soluble CD44-immunoglobulin fusion protein as well as by anti-CD44 antibody, and to depend on the presence of the cytoplasmic domain of CD44. However, cells expressing CD44H cytoplasmic deletion mutants retain significant binding capacity to hyaluronate-coated substrate. Taken together, our results provide direct evidence that CD44H plays a major role in regulating cell migration on hyaluronate-coated substrate.

Actin-binding protein requirement for cortical stability and efficient locomotion.

Three unrelated tumor cell lines derived from human malignant melanomas lack actin-binding protein (ABP), which cross-links actin filaments in vitro and connects these filaments to plasma membrane glycoproteins. The ABP-deficient cells have impaired locomotion and display circumferential blebbing of the plasma membrane. Expression of ABP in one of the lines after transfection restored translocational motility and reduced membrane blebbing. These findings establish that ABP functions to stabilize cortical actin in vivo and is required for efficient cell locomotion.

Growth inhibition of murine melanoma and human colon carcinoma by recombinant human platelet factor 4.

Although it is well established that angiogenesis is essential to tumor development, no human protein with high specificity and efficacy for prevention of angiogenesis has been characterized. In a previous study, we demonstrated that recombinant platelet factor 4 (rPF 4) inhibited angiogenesis in the chicken chorioallantoic membrane. In the present study, we have extended that finding to the use of recombinant human platelet factor 4 (rHuPF 4) to inhibit solid tumor growth in the mouse. rHuPF 4 effectively suppressed the growth of the B16-F10 murine melanoma in syngeneic C57BL/6J hosts and prevented the growth of primary tumors of both B16-F10 murine melanoma and HCT 116 human colon carcinoma in semisyngeneic CByB6F1/J female athymic nude mice. These two transformed cell lines were completely insensitive to rHuPF 4 in vitro at levels (50 micrograms/mL) that extensively inhibit normal endothelial cell proliferation. The migration of human endothelial cells was also inhibited at these concentrations of rHuPF 4, suggesting a second mechanism by which rHuPF 4 may modulate capillary development. The observed antitumor effects of rHuPF 4 might be due to the inhibition of angiogenesis. This finding could have implications for the development of novel therapeutic approaches to angiogenic diseases. Alternative, and possibly concurrent, mechanisms of the rHuPF 4 antitumor effect include lymphokine-activated killer cell activation and the induction of other cytokines.

Expression of a transfected H-2Kb gene in B16 cells correlates with suppression of liver metastases in triple immunodeficient mice.

In vivo experiments performed with NIH (nu/nu, bg/bg, xid/xid) triple immunodeficient (TD) mice revealed the striking ability of i.v. injected B16-F1 and B16-F10 murine melanoma cells to colonize not only the lungs but also the liver of TD mice. Subsequently, B16 melanoma cell cultures, which express very low levels of H-2Kb antigen, were cotransfected with plasmids pRSVneo, containing the neomycin resistance gene, and 6-2B1pMT, expressing the H-2Kb complentary DNA under the control of the metallothionein enhancer-promoter. Several neomycin-resistant clones were analyzed for H-2Kb and H-2Db expression by RNase protection and flow cytometry assays. All parental lines and transfected clones expressed normal levels of H-2Db mRNA, while only some of the transfected clones expressed easily detectable levels of H-2Kb mRNA. Moreover, in these clones H-2Kb expression could be enhanced in the presence of Zn2+, indicating that the metallothionein enhancer was functioning properly. Parental cells and transfected clones were injected i.v. in TD mice to assess the possible involvement of H-2Kb antigen in regulating the metastatic potential of B16 melanoma cells. We observed a remarkable correlation between expression of H-2Kb antigen and suppression of liver-specific metastases in TD mice. Identical results were obtained when we gave TD mice injections of mixed populations of transfectants expressing H-2Kb antigen, obtained by fluorescence-activated cell sorting. These experiments allowed us to rule out the possibility that the observed changes in metastatic potential were due to clonal variability among individual transfected clones. Taken together, the results of our in vivo studies with immunodeficient mice support the notion that specific major histocompatibility complex Class I molecules modulate the metastatic potential of malignant cells also by mechanisms which are independent of their well-established role in antigen presentation.

Prevention of human prostate tumor metastasis in athymic mice by antisense targeting of human angiogenin.

PURPOSE: Angiogenin is a potent positive mediator of neovascularization, a process required for both primary tumor growth and metastasis. In the present study, the effect of a fully phosphorothioated antisense oligodeoxynucleotide, designated JF2S, targeting the AUG translation initiation codon region of human angiogenin, on human prostate tumor development and metastasis in athymic mice was examined. EXPERIMENTAL DESIGN: JF2S was evaluated for its capacity to affect in vitro synthesis of angiogenin and subsequent tumorigenicity of transiently transfected prostate tumor cells in mice. In vivo treatment experiments were then conducted in which JF2S was used to prevent formation of tumors in an ectopic model and metastasis in an orthotopic model. RESULTS: Transient transfection of tumor cells with JF2S inhibited both angiogenin gene expression in vitro and tumorigenicity of these transfected cells in athymic mice. In therapy experiments, local treatment with JF2S completely protected mice from developing prostate tumors after s.c. injection of PC-3 human prostate tumor cells (P < 0.0001, survivor analysis). Most importantly, systemic prophylactic administration of JF2S prevented, in 47% of mice, formation of regional iliac lymph node micrometastases arising from primary tumors growing in the more natural orthotopic prostate setting (P = 0.0003, Fisher's exact test). Furthermore, total protection from regional metastasis occurred in those mice in which JF2S treatment successfully diminished human angiogenin expression in vivo. Tumor-associated angiogenesis was also impaired by JF2S treatment. When therapy was delayed until all of the mice harbored primary tumors in the prostate, the incidence of regional metastasis was still significantly decreased (P < 0.005, survivor analysis). CONCLUSIONS: These findings demonstrate that human prostate cancer establishment and spread in athymic mice is extremely susceptible to targeted disruption of tumor-derived human angiogenin gene expression. Therefore, angiogenin is a valid target against which to devise preventative strategies for prostate cancer metastasis.

Role of integrin alpha 2 beta 1 (VLA-2) in the migration of human melanoma cells on laminin and type IV collagen.

The random cell migration of four human melanoma cell lines on laminin and type IV collagen-coated substrates was studied by video time-lapse image analysis and compared to the expression of a number of beta 1 integrins including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 1 using flow cytometry. These integrins were heterogeneously expressed in the four cell lines tested with three of four lines expressing alpha 2 beta 1. The melanoma cell line that did not express alpha 2 beta 1 exhibited weak attachment and low cell migration rate on both laminin and type IV collagen, whereas the other melanoma cell lines showed an increase in attachment and mean cell migration rate in a dose-dependent manner on the matrix molecules (p < 0.001). The enhanced migration seen in the three cell lines could be specifically inhibited by function blocking anti-beta 1 and anti-alpha 2 monoclonal antibodies (p < 0.001) but not by function blocking anti-alpha 3 and anti-alpha 6 monoclonal antibodies. Image analysis of the cells before and after treatment with anti-beta 1 and anti-alpha 2 MoAb indicated that the inhibition of migration did not result in detectable cell detachment, retraction of cell processes, or other significant cell-shape change. Taken together, the findings suggest that the observable enhanced migration on laminin and type IV collagen of a number of human melanoma cell lines is largely mediated by integrin alpha 2 beta 1.

Migration of human melanoma cells on hyaluronate is related to CD44 expression.

Phenotypic and functional aspects of melanoma-hyaluronate interactions were investigated by studying the expression of CD44, cell migration, and transmembrane penetration of human melanoma cell lines on hyaluronate-coated substrates. Expression of CD44 was tested by flow cytometry on seven human melanoma cell lines. Strong reactivity with anti-CD44 monoclonal antibody was observed in four of seven of the cell lines. Migration studies of CD44(+) cell lines on hyaluronic acid- and chondroitin-6-sulfate-coated substrates, using time-lapse video-microscopy, showed a dramatic dose-dependent increase in migration rate on hyaluronate but not on chondroitin-6-sulfate. Moreover, CD44(-) cell lines showed no modification in migration rate on either substrate. Addition of soluble hyaluronate produced a dose-dependent inhibition of acceleration of CD44(+)cells on hyaluronate-coated substrates, whereas addition of chondroitin-6-sulfate had no effect. Migration inhibition experiments with soluble CD44 (CD44 receptor globulin) also showed specific blocking of the migration of CD44(+) cells on hyaluronate. Haptotactic invasion was increased in CD44(+) cell lines through hyaluronate-coated polycarbonate membranes, whereas no change was detected on chondroitin-6-sulfate-coated membranes. CD44(-) cell lines showed no response to either type of coating. In the melanoma cell lines tested, the expression of CD44 correlated with in vitro migration and invasiveness on hyaluronate substrates. Taken together, our data are consistent with the suggestion that CD44 may play a role in stimulating in vivo aggressiveness of tumors through hyaluronate-rich stroma.

Focal adhesion kinase (p125FAK) expression correlates with motility of human melanoma cell lines.

Focal adhesion kinase (p125FAK), a recently characterized protein localized within focal adhesion plaques, is believed to play a role in extracellular matrix-integrin-mediated signal transduction involving cytoskeletal proteins. We studied p125FAK expression, distribution, and relation to cell migration in six human melanoma cell lines. Western blot analysis detected differential expression of p125FAK among these lines that was directly proportional to the amount of phosphorylated p125FAK. Time-lapse image analysis of the cell lines exhibited 10-fold differences in the mean migration rates on fibronectin-coated substrates. Regression analysis revealed that p125FAK expression correlated significantly with mean migration rate in the six melanoma lines tested. Double immunofluorescent labeling for p125FAK and actin in these lines demonstrated p125FAK plaques that were localized to actin stress-fiber termination sites in the periphery of cells. The number of p125FAK plaques in the melanoma cell lines was heterogeneous, but the cell lines with more p125FAK plaques per cell exhibited significantly higher mean migration rates on fibronectin as compared with cell lines with fewer p125FAK plaques per cell. The findings support the hypothesis that focal adhesion tyrosine kinase modulates cytoskeletal function during melanoma cell migration on fibronectin.

Role of cytoplasmic dynein in melanosome transport in human melanocytes.

Cytoplasmic dynein is a microtubule-associated retrograde-directed motor molecule for transport of membrane-bound organelles. To determine whether cytoplasmic dynein is expressed in melanocytes, we performed reverse transcriptase polymerase chain reaction using melanocyte cDNA and primers complementary to human brain cytoplasmic dynein heavy chain. A polymerase chain reaction product of the expected molecular size was generated and the identity was confirmed by sequence analysis. Western blotting of total melanocyte proteins reacted with an anti-intermediate chain cytoplasmic dynein antibody identified the appropriate 74 kDa band. To determine whether cytoplasmic dynein plays a role in melanosome transport, duplicate cultures were treated with cytoplasmic dynein antisense or sense (control) oligodeoxynucleotides and the cells were observed by high-resolution time-lapse microscopy, which allows visualization of melanosomal aggregates and individual melanosomes. Antisense-treated melanocytes demonstrated a strong anterograde transport of melanosomes from the cell body into the dendrites, whereas melanosome distribution was not affected in sense-treated melanocytes. To determine whether ultraviolet irradiation modifies cytoplasmic dynein expression, melanocyte cultures were exposed to increasing doses of solar-simulated irradiation, equivalent to a mild to moderate sunburn exposure for intact skin. Within 24 h, doses of 5 and 10 mJ per cm2 induced cytoplasmic dynein protein, whereas doses of 30 mJ per cm2 or more were associated with decreased levels of cytoplasmic dynein compared with sham-irradiated controls. Our data show that cytoplasmic dynein participates in retrograde melanosomal transport in human melanocytes and suggest that the altered melanosomal distribution in skin after sun exposure is due, at least in part, to decreased cytoplasmic dynein levels resulting in augmented anterograde transport.

Zyxin redistributes without upregulation in migrating human keratinocytes during wound healing.

Cell migration, growth, and survival is modulated by focal adhesions linking extracellular matrix proteins, cell adhesion molecules, and the cytoskeleton. Zyxin is a focal adhesion phosphoprotein that shares homology with Listeria ActA protein in promoting actin filament assembly; it also has specialized protein-protein interface domains implicating an important role in cell growth and differentiation. We investigated the distribution of zyxin in normal and migrating human keratinocytes in wounds in vitro and in situ using confocal laser microscopy. Zyxin expression in high-density nonmigrating keratinocytes versus low-density migrating keratinocytes was determined by western immunoblotting and time lapse image analysis. In normal epidermis, zyxin exhibited a punctate staining pattern throughout the cytoplasm and was excluded from the intercellular spaces. In wounds, the punctate staining also localized in the edge of the migrating keratinocyte sheets; however, intercellular spaces were absent. Likewise, in vitro keratinocytes showed punctate staining throughout the cytoplasm. Migrating cultured keratinocytes next to wounds, however, had large focal contacts in the cell periphery where actin bundles converged at focal adhesions. Western immunoblots and confocal experiments with protein synthesis inhibition by cycloheximide confirmed that this difference in distribution of zyxin in migrating versus nonmigrating keratinocytes is due to the redistribution and not upregulation of zyxin. The abundance of zyxin and its relative change in distribution from normal to migrating keratinocytes in wounds is consistent with its role in cytoskeletal organization of actin bundles.

Comparison of acral nevomelanocytic proliferations in Japanese and whites.

The preponderance of malignant melanomas that occur in the Japanese population affect the palmar and plantar areas, in contrast to those of whites, which occur predominantly on the head, neck, and trunk. Furthermore, a precursor lesion, the dysplastic nevus, has been well defined in nonacral white skin but not in the Japanese. In this paper, a similarity between benign and atypical acral nevomelanocytic proliferations in Japanese and whites is described. However, it appears that in the Japanese group the melanocytes in benign and atypical proliferations are less dendritic than those noted in whites, and, in both groups, extensive pigmentation was noted in both the epidermal and dermal components of benign and malignant lesions in this limited review. A practical approach to classifications of atypism is, likewise, offered.

Differential effects of interleukin 1-alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) on motility of human melanoma cell lines on fibronectin.

Interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) induce a motogenic response in a number of benign and malignant cells. We examined the chemokinetic effects of these cytokines on the cell migration of four melanoma cell lines on fibronectin using modified Boyden chambers and video-time lapse analysis. Flow cytometry analysis of IL-1 receptors, TNF receptors, and shifts in beta 1 integrin expression were correlated with the effects of these cytokines on cell migration on fibronectin. The four melanoma cell lines exhibited heterogeneous expression of types I and II IL-1 receptors as well as p60 TNF receptors. Scant p80 TNF receptor expression was detected on only one cell line. Three of four melanoma cell lines demonstrated type I IL-1 receptors by Western blotting. IL-1 alpha and TNF-alpha induced heterogeneous modulation of beta 1 integrin expression in the four melanoma cell lines tested; downward shift of the alpha 2, alpha 3, alpha 4, and beta 1 integrin subunits was detected among three of the melanoma cell lines as were upward shifts of the alpha 4, alpha 5, and alpha 6 integrin subunits among three of the melanoma cell lines. IL-1 alpha and TNF-alpha induced enhanced migration on fibronectin in one of the melanoma cell lines and were related to an upward shift in the alpha 4 and alpha 5 integrin subunit expression. Taken together, the findings indicate that expression of a particular receptor for IL-1 or TNF does not necessarily signal a motogenic response in melanoma cells, but induces heterogeneous shifts in beta 1 integrin expression. However, upregulation in alpha 4 and alpha 5 integrin subunits appears to relate to enhanced migration on fibronectin.

Histopathologic recognition and grading of dysplastic melanocytic nevi: an interobserver agreement study.

Before the controversies surrounding dysplastic melanocytic nevi are resolved, dermatopathologists must be able to reliably distinguish dysplastic nevi from common acquired nevi and malignant melanoma. To establish whether grading of melanocytic dysplasia has any biologic relevance, dermatopathologists must be able to consistently recognize two or more grades of atypia. We studied the concordance among five dermatopathologists for recognition and grading of 60 nevomelanocytic lesions. Ten cases from each of the following categories of melanocytic proliferation were retrieved from the Massachusetts General Hospital files: 1) common melanocytic nevi, 2) melanocytic nevi with features of dysplastic nevi, 3) dysplastic nevi with slight cytologic atypia, 4) dysplastic nevi with moderate cytologic atypia, 5) dysplastic nevi with severe cytologic atypia, and 6) primary malignant melanoma. The slides were reviewed independently; no discussion of diagnostic criteria preceded the review. Overall concordance for diagnosing dysplastic nevi was 77%, with a kappa statistic of 0.55-0.84. Furthermore, in grading dysplastic nevi, experienced dermatopathologists had a concordance ranging from 35% to 58% (kappa value 0.38-0.47). Those with less experience in grading dysplastic nevi had a concordance of 16-65% (kappa value 0.05-0.24). The five observers in this study reliably distinguished dysplastic nevi from common acquired nevi and malignant melanoma. Further refinement of the criteria for grading of nevo-melanocytic dysplasia and experience in grading are critical for accuracy in subcategorization of dysplastic nevi. Consistent, reproducible subcategorization of dysplastic nevi is a requisite before the issue of biologic or prognostic relevance of grading (dysplastic nevi) can be addressed. This study supports the validity of existing criteria for the diagnosis of dysplastic nevi because the problems in diagnosis were at the limits of the spectrum, namely, discrimination of slightly atypical dysplastic nevi from common nevi and severely atypical dysplastic nevi from radial growth phase melanoma.

Kinesin participates in melanosomal movement along melanocyte dendrites.

Movement of melanosomes along melanocyte dendrites is necessary for the transfer of melanin pigment from melanocytes to basal and suprabasal keratinocytes, an event critical to epidermal photoprotection and maintenance of normal skin color. Recent murine data suggest that in melanocyte dendrites the microtubule-associated melanosome movement is bidirectional and that actin-associated myosin V secures the peripheral melanosomes, preparing them to be transferred to surrounding keratinocytes. We now report that human melanocytes express high levels of kinesin, a molecule that participates in microtubule-associated transport of organelles in other cell types, and that ultrastructurally kinesin molecules are closely associated with melanosomes. To determine whether kinesin participates in melanosomal transport, cultured melanocytes were treated with sense or antisense oligonucleotides complementary to kinesin heavy chain sequences. Antisense oligonucleotides decreased kinesin protein levels and inhibited the bidirectional movement of the melanosomes, promoting their backward movement. Furthermore, guinea pigs were exposed to ultraviolet B irradiation, known to enhance transport of melanosomes from melanocytes to epidermal keratinocytes, and then were treated with kinesin sense or antisense oligonucleotides. The areas that were treated with kinesin antisense oligonucleotides showed significantly less pigmentation clinically and histologically than control (sense) oligonucleotide-treated areas. As observed ultrastructurally, in antisense-treated areas melanosomes remained in melanocyte dendrites but over several days were not transferred to the surrounding keratinocytes. Our study supports a major role for kinesin in microtubule-associated anterograde melanosomal transport in human melanocyte dendrites.

Selective expression of FLIP in malignant melanocytic skin lesions.

FLIP (FLICE Inhibitory Protein) is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits cell death mediated by all death receptors including Fas and TRAIL. FLIP has recently been shown to favor tumor growth and immune escape in mouse tumor models. We analyzed FLIP expression by immunohistochemistry in a panel of 61 benign and malignant human melanocytic skin lesions. FLIP expression was undetectable in all but one benign melanocytic lesion (31/32, 97%). In contrast, FLIP was strongly expressed in most melanomas (24/29 = 83%). Overexpression of FLIP by transfection in a Fas- and TRAIL-sensitive human melanoma cell line rendered this cell line more resistant to death mediated by both TRAIL and FasL. Selective expression of FLIP by human melanomas may confer in vivo resistance to FasL and TRAIL, thus representing an additional mechanism by which melanoma cells escape immune destruction.

Actin-binding protein expression in benign and malignant melanocytic proliferations.

Studies on melanoma cell lines indicate the expression of actin-binding protein (ABP), a peripheral cytoplasmic protein that crosslinks actin, is important for melanoma cell motility. We used an ABP-specific monoclonal antibody to characterize ABP expression in 18 benign nevi and 28 primary and metastatic malignant melanomas. Heterogeneous expression of ABP staining was observed in metastatic melanoma. No clear differences in ABP staining were identified among compound nevi, dysplastic nevi, and superficial spreading melanoma; however, the lentiginous intraepidermal component of the benign and malignant lesions and the pagetoid cells of superficial spreading malignant melanoma were negative for ABP. In contrast, the nested intraepidermal and dermal components of both benign nevi and primary malignant melanoma were positive. The differential expression of ABP of the lentiginous component as opposed to the intraepidermal nests and pagetoid cells of benign nevi or melanoma may represent a capacity of the nested melanocytes to migrate from the epidermis to the dermis during maturation or invasion. Taken together, the findings support that ABP may be important for cell-cell adhesion during tumorigenesis and may play a role in tumor cell ameboid motility during tissue invasion.

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.