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Expression of proliferating cell nuclear antigen in Spitz nevus
Abstract
Epithelioid and spindle cell nevus (ESN; Spitz nevus) is a histologically well-described entity. We hypothesized that the features of ESN may reflect activation by a proliferative stimulus.
Nevocytes and keratinocytes in ESN and control specimens were examined for expression of the proliferation marker, proliferating cell nuclear antigen (PCNA).
Standard immunohistochemical methods were used to examine the expression of PCNA in a series of ESN, other nevi, and malignant melanoma.
PCNA was detected in nevocytes in a significant percentage of ESN but not in other nevi. PCNA expression was increased in basilar keratinocytes in ESN when compared with staining of basilar keratinocytes in normal epidermis. In other melanocytic nevi and noninflamed melanoma, PCNA expression in keratinocytes was similar to that in normal control tissue.
Increased PCNA labeling in nevocytes and keratinocytes in ESN suggests that a growth stimulus, present within some of these lesions, affects both keratinocytes and nevocytes.
... Various monoclonal antibodies against the Ki-67 nuclear antigen have been used to measure the proliferative index (PI) in malignant melanomas, and correlations have been drawn between that analyte and tumor thickness, metastatic potential, or overall prognosis (18 -20). Fewer studies have applied PI as a discriminative tool in separating melanocytic nevi from malignant melanomas, and, with some exceptions, most of those analyses were based on a limited number of cases or they used dissimilar methods, some of which would be impractical in routine diagnostic pathology (6,(21)(22)(23)(24)(25). Our goals in part reproduced those of earlier assessments of PI in melanocytic lesions, but they were also somewhat different. ...
Ki-67 labeling of paraffin sections has been correlated with the number of cells in non-G(o) phases of the replicative cell cycle, and this immunohistochemical technique has been applied to the evaluation of a variety of human neoplasms. Similarly, immunolabeling for p53 protein has been used to detect mutations in the corresponding gene, as a reflection of possible cellular transformation in the same context. Both of these techniques were applied to 253 melanocytic tumors of the skin to assess their possible utility in the diagnosis and subcategorization of such lesions. They included 76 banal (common) nevi (CN), 39 Spitz nevi (SN), 62 superficial spreading malignant melanomas in radial growth (SSMMs), 32 nodular malignant melanomas (NMMs), 21 lentigo maligna melanomas in radial growth (LMMs), and 23 melanomas arising in association with preexisting compound nevi (MCN). One hundred cells were counted randomly in each tumor, and dark, exclusively nuclear reactivity was scored as positive labeling; results were recorded as percentages. Negligible Ki-67 and p53 labeling was seen in CN and SN, at a level that was similar to that obtained in cases of LMM and MCN. The largest proportion of Ki-67-positive and p53-positive cells was observed in NMMs, followed by SSMMs. Radial growth-phase SSMMs and LMMs demonstrated immunoprofiles that were similar to those of melanocytic nevi, and MCN did so as well. The prototypical malignant melanocytic tumor representing the vertical growth phase-nodular melanoma--demonstrated a statistically significant difference from all other lesions in this study with respect to Ki-67 index (P = .008, chi2) and p53 reactivity (P < .000001, chi2). Subsequent concurrent use of a Ki-67 threshold index of 10% and a p53 index of 5% correctly indicated the presence of vertical growth in 75% of NMMs, whereas only 8% of radial growth phase melanomas of other types were colabeled at the same levels of reactivity for the two markers (P < .00001, chi2). Thus, although the distinction between benign and malignant melanocytic tumors could and should not be based on immunohistology for Ki-67 and p53, these results suggest that the latter determinants may, in fact, be used as an adjunct to morphology in the recognition of the vertical growth phase in cutaneous malignant melanomas.
... Proliferation cell nuclear antigen (PCNA), a polymerase accessory protein, is present in the cell cycle, peaking at the G1/S phase (74). Many studies have shown a correlation between increased melanocytic tumor cell PCNA staining and malignancy (20,(74)(75)(76)(77). Kanoko et al. (20) showed that PCNA immunostaining is strongest in metastatic melanoma. ...
We critically reviewed recent literature reports of 25 melanocytic immunohistochemical markers. This review organizes and summarizes the many new studies of old and novel melanocytic markers and identifies the most promising diagnostic immunohistochemical markers that can be used to distinguish melanocytic from nonmelanocytic lesions and benign melanocytic from malignant melanocytic lesions.
... The PCNA index, as defined by the number of positive staining cells per 1000 tumor cells, is different in benign nevi vs melanoma, averaging 7.2 in nevi and 248.5 in malignant melanoma. 66 In one study, the PCNA index proved more efficacious than the Breslow depth in predicting locoregional recurrences and metastases. 67 The predictive value of Ki-67 correlated more closely in one study of uveal melanoma with S phase, shorter survival, histological type, and tumor size than did PCNA expression. ...
Since the 1960s, the clinical characteristics of melanoma, its histopathology and its biological basis have been the subject of intense study at pigmented lesion clinics in North America, Europe, and Australia. More recently, the immense database of the Melanoma Committee of the American Joint Committee on Cancer (AJCC) has been exploited through complex mathematical models to measure the impact of various histologic features of primary melanomas and of sentinel lymph node deposits and to correlate these parameters with patient survival. The wealth of modern information available to pathologists and clinicians has become of vital interest to the prognostication of the individual patient with melanoma. The purpose of this review is to bring to the attention of anatomic pathologists the essential characteristics of the pathology report for primary cutaneous melanoma in the modern era.
Recent advances in our understanding of the biology of melanoma have focused on the nature of disease initiation and progression. With respect to the former, the genetics of melanoma, and in particular the impact of genetic defects on dysregulation of the cell cycle, are discussed in this chapter. With respect to the latter, the chapter discusses the acquisition of growth factor autonomy by neoplastic clones, the acquisition of capabilities for invasion and metastasis from the standpoint of cell adhesion, motility, and matrix digestion, and the nature of the immunologic response to melanoma. This chapter provides an update on the recent advances in our comprehension of melanomagenesis from advances in molecular pathology. The p53 gene encodes a 53-kDa nuclear phosphoprotein that is a member of a group of proteins that regulates the cell cycle.
Atypical cutaneous melanocytic lesions, including those with Spitzoid features, can be difficult to categorize as benign or malignant. This can lead to suboptimal management, with potential adverse patient outcomes. Recent studies have enhanced knowledge of the molecular and genetic biology of these lesions and, combined with clinicopathological findings, is further defining their biological spectrum, classification, and behavior. Sentinel node biopsy provides important prognostic information in patients with cutaneous melanoma, but its role in the management of melanocytic lesions of uncertain malignant potential (MELTUMP) is controversial. This paper examines the role of molecular testing and sentinel node biopsy in MELTUMPs, particularly atypical Spitzoid tumors.
Mutations in the p53 tumor suppressor gene, located at chromosomal locus 17pI3, are the most commonly seen genetic alterations found in human malignancies. Their role in the pathogenesis of malignant melanoma is thought to be limited, although variable results have been reported in reference to immunoreactivity for putatively mutant p53 protein (mp53) in melanocytic lesions in general. In that light, the authors undertook an immunohistologic evaluation of 256 well-characterized tumors in that category, including common nevi (CN; n=73); Spitz nevi (SN; n=40); nodular melanomas (NMMs; n=32), superficial spreading melanomas (SMMs; n=65); lentigo maligna melanomas (LMMs; n=23); and melanomas arising in preexisting nevi (MANs; n=23). One hundred cells were counted manually in randomly selected high-power microscopic fields, in regard to nuclear labeling for mp53. Results were recorded semiquantitatively, as negative, positive (1-4% of tumor cells); and positive (>5% of tumor cells). No examples of CN or SN demonstrated any immunoreactivity whatever for mp53, whereas 105 of 143 melanomas (73%) did so. However, an mp53 index of >50% was seen in only 29% of the latter lesions. NMMs were most often mp53-positive and showed the highest numerical level of nuclear labeling, followed in respective order by SMMs, and LMMs/MANs. These results suggest that negative mp53-immunostaining cannot be equated with the diagnostic interpretation of a benign melanocytic neoplasm, because ²⁷% of melanomas also failed to label for that determinant. However, the presence of mp53-immunolabeling in a melanocytic proliferation-even if at low levels-should conversely prompt careful consideration of melanoma as the favored diagnosis in the confined setting of morphologically difficult cases, inasmuch as no example of CN or SN in this series had that characteristic.
Aims:
The differential diagnosis between Spitz naevus and spitzoid melanoma can be extremely difficult, or even impossible. In recent years, many attempts have been made to find specific histopathological or immunohistochemical markers, although none has proved successful. Because the prognosis and treatment of each are very different, it is important to distinguish between these entities. We evaluated the ability of the fluorescence in situ hybridization (FISH) assay-designed to detect the copy number of the RREB1 (6p25), MYB (6q23) and CCND1 (11q13) genes and of centromere 6 (Cep 6)-in order to distinguish between Spitz naevus and spitzoid melanoma.
Methods and results:
We evaluated 12 spitzoid melanomas and six Spitz naevi from our records. The diagnosis of both conditions was based on previously described histopathological criteria. We obtained valuable results for FISH in eight spitzoid melanomas and five Spitz naevi. Chromosomal aberrations were detected in seven of the eight spitzoid melanomas (FISH-positive) and in none of the five Spitz naevi. The FISH-negative spitzoid melanoma was the least typical in its group.
Conclusions:
FISH was able to distinguish between Spitz naevus and spitzoid melanoma, with a sensitivity of 87.5% and a specificity of 100%. Our findings suggest that FISH could prove a useful tool in the differential diagnosis between these entities.
Introduction: The term spitzoid melanoma (SM) is reserved for a rare group of tumors with striking resemblance to Spitz nevus, often developing in children diagnosed in retrospect after the development of metastases.
Objectives: To determine the biological significance of SM and to analyze the effectiveness of adjuvant diagnostic techniques.
Materials and methods: A retrospective, observational study of 38 cases of SM in patients younger than 18 years. Histological type, Clark level and Breslow thickness, radial and vertical growth phase, mitotic count/mm2, ulceration, regression, vascular and perineural invasion, satellitosis, cytology and associated nevi were reviewed. An immunohistochemical analysis with HMB45 and Ki67 was performed in 10 cases. These features were correlated to patient’s stage and outcome.
Results: Analysis of histological and immunohistochemical features should allow accurate diagnosis in most cases. Given the low mortality rate, no conclusions about the prognostic significance of histological parameters of the primary tumor could be established.
Conclusion: We report the largest series of SM from a unique center. Although these patients may have a better prognosis than adults, some patients with SM develop metastasis and die, particularly after age 11 years. Therefore, we recommend using the same treatments as in adults.
The last two decades have seen spectacular advances in our understanding of the biology of melanoma and, in particular, the
mechanisms operative in disease initiation and progression. In disease initiation, the genetics of melanoma, and, in particular,
the impact of genetic defects on dysregulation of the cell cycle, are key issues in malignant transformation and a major focus
of this chapter. Regarding disease progression, consideration is also given herein to the acquisition of growth factor autonomy
by neoplastic clones and to the acquisition of capabilities for invasion and metastasis from the standpoint of cell adhesion,
motility, and matrix digestion. These events have specific morphological correlates which will be discussed briefly.
An appropriate biopsy is the pivotal procedure that facilitates accurate histopathologic diagnosis of a pigmented skin lesion. Excisional skin biopsy is the method of choice for removing a suspected malignant melanoma. More than 95% of malignant melanomas that involve the skin belong to one of the four most common clinicopathologic categories: superficial spreading, nodular, lentigo maligna, and acral lentiginous melanoma. A small but important group of cutaneous melanomas can be classified as unusual variants. Many of these unusual variants have a distinct histopathologic appearance; they include desmoplastic melanoma, neurotropic melanoma, pedunculated melanoma, metastatic melanoma, amelanotic melanoma, melanoma arising within a benign nevus, regressing ("invisible") melanoma, and balloon cell melanoma. Other lesions may simulate malignant melanoma histopathologically. Immunohistochemical stains, such as S-100 protein, vimentin, keratin, and HMB-45, are useful for distinguishing these lesions from true melanoma.
Recent evidence has implicated cyclins and cyclin-dependent kinases in the evolution and progression of various malignancies. We studied the immunohistochemical expression of cyclin A, cyclin B, and cyclin-dependent kinase p34cdc2 in a broad spectrum of benign and malignant melanocytic lesions. Formalin-embedded, parrafin-fixed tissue sections from 66 malignant melanomas (MM) and 60 benign nevi were examined for the expression of these cell-cycle proteins. The results were compared with the standard proliferative marker Ki-67 and mitotic index. MM showed significantly higher immunoreactivity for cyclin A, cyclin B, p34cdc2, and Ki-67 compared with benign nevi. Cyclin A, p34cdc2, and Ki-67 displayed strong co-expression in MM. Overexpression of cyclin A and p34cdc2 correlated with histological type, mitotic activity, Ki-67 index, tumor thickness, Clark's level, and clinical outcome in MM. In invasive MM, increased immunostaining of cyclin A and Ki-67 were associated with decreased patient survival. These findings indicate potential roles of mitotic cyclins and cyclin-dependent kinases in the pathogenesis and progression of malignant melanoma.
Spitz nevi are benign melanocytic neoplasms of children and young adults that can be exceedingly difficult to distinguish from malignant melanomas. Although a nearly definite diagnosis can be made in most cases, the histological distinction between Spitz nevi and melanomas is equivocal in about 6% to 8% of cases. In those cases, and perhaps even with presumed benign Spitz nevi, clear surgical margins are desirable. The most helpful differentiating features of Spitz nevi are patient age, sharp demarcation, symmetry, maturation of melanocytes at the base, and epithelial hyperplasia. None of these criteria are completely reliable, and multiple other criteria must be considered as well.
The histopathologic differential diagnosis of Spitz nevus (SN) from malignant melanoma (MM) may be difficult.
Our purpose was to determine the staining pattern and usefulness of MIB-1 antibody, which recognizes Ki-67 antigen in formalin-fixed, paraffin-embedded tissue, as an adjunct to the histopathologic differential diagnosis of SN.
Twenty-five compound SNs, 27 MMs, and 26 compound nondysplastic melanocytic nevi (MNs) were immunostained with the MIB-1 antibody.
The mean counts of MIB-1--stained tumor cells of the epidermal and dermal components, both alone and together, were significantly lower in SNs and MNs than in MMs (P <.0001). The dermal counts showed the best discriminating power. In addition, the mean dermal/epidermal count ratios for MIB-1 in SNs and MNs (0.25 and 0.23, respectively) were significantly lower than the corresponding ratio (0.94) in MMs (P <.0001).
MIB-1-stained tumor cell counts, especially of the dermal component, and dermal/epidermal MIB-1 count ratios may be helpful as an adjunct to the histopathologic differential diagnosis of SN.
Melanomas can be difficult to diagnose histologically if they deviate in their growth pattern or cytology only minimally from a nevus. On occasion, even experts on melanocytic lesions may not reach a consensus on whether a lesion is a benign but unusual nevus or a malignant melanoma mimicking a nevus. This diagnostic dilemma is particularly well known for the distinction of Spitz nevus from melanoma. Diagnostic uncertainty and disagreement among consultant pathologists lead to confusion about the prognosis and clinical management of patients. In this study we present the clinical and pathologic findings of 10 patients with diagnostically controversial melanocytic tumors, who underwent sentinel lymph node biopsy. In all of these cases, the diagnostic controversy among experts was between Spitz nevus and melanoma. Seven patients were female, and three were male, ranging in age from 7 to 46 years (mean 21 years). Histologic examination of the sentinel lymph nodes revealed tumor deposits in the lymph node parenchyma in 5 of 10 patients. Among patients with positive sentinel lymph nodes, two had satellite nodules and one showed additional tumor deposits in three nonsentinel regional lymph nodes. All patients are alive and free of disease with a follow-up of 10-54 months (mean 34 months). Our study illustrates the role of a sentinel lymph node biopsy in the evaluation of patients with diagnostically controversial melanocytic tumors. Although the presence of metastatic tumor deposits in the sentinel lymph node supports the diagnosis of malignant melanoma, further studies are needed to determine the prognostic significance of the sentinel lymph node findings in such patients.
The Spitz nevus is a benign melanocytic lesion that can be identified reliably in many cases by conventional histopathological criteria. However, there are subsets of Spitz nevi and of malignant melanoma that closely resemble each other and represent diagnostic challenges. S100 proteins are of interest because of their involvement in neoplastic processes and their genes are clustered in chromosome 1q21. Chromosome 1 contains mutations in several types of tumors, including melanomas. The expression of different S100 proteins (A2, A6 and A8/A9 or A12) was examined in 42 Spitz nevi, 105 melanomas, and 73 melanocytic nevi to test the hypothesis that their expression differs among these entities and may contribute to the distinction between these entities. The results showed an up-regulation of S100A6 protein in Spitz nevi, melanomas, and melanocytic nevi but with a different percentage of positivity and pattern of immunoreactivity. The differences between these three entities were statistically significant (P <.001). All 42 Spitz nevi (100%) showed strong and diffuse S100A6 protein expression, both in junctional and in dermal components of the nevi. Thirty-three percent of melanomas expressed S100A6 (35/105). The expression was mainly weak (30/35) and patchy in the dermal component and was negative or minimal in the junctional component. Fifty-six percent of different subtypes of melanocytic nevi (41/73) expressed S100A6, almost all of them weakly (40/41) and in the dermal component. Normal intraepidermal melanocytes were negative. The melanocytic cells in these three entities did not express S100A2, S100A8/A9 or A12. However, an up-regulation of S100A2 and S100A8/A9 or A12 proteins was observed in normal keratinocytes in the epidermis overlying Spitz nevi and melanomas, without differences. In summary, a simple immunohistochemical test for S100A6 protein differentiated between Spitz nevi, melanomas, and melanocytic nevi. This marker could be used when the distinction is very difficult or controversial in routine studies, especially when there is a junctional component. Further molecular analyses of the S100A6 protein and gene should be performed to study the underlying genetic bases for such differences.
A subset of Spitz nevi poses substantial diagnostic difficulty, even among experts, due to its resemblance to malignant melanoma. These lesions are termed atypical Spitz nevi/tumors and there is currently a lack of objective criteria for predicting their biologic behavior. We compared the expression of Ki-67, p21, and fatty acid synthase by immunohistochemistry in 10 atypical Spitz nevi, 28 typical Spitz nevi, 19 compound melanocytic nevi and 18 invasive malignant melanomas. There was a progressive increase in fatty acid synthase cytoplasmic expression with statistically significant differences observed between Spitz nevi and atypical Spitz nevi (P=0.003) and between atypical Spitz nevi and malignant melanoma (P<0.050). Ki-67 nuclear staining was lower in both typical and atypical forms of Spitz lesions than in malignant melanoma (P<0.001). The degree of P21 nuclear expression in atypical Spitz nevi was not significantly different than in Spitz nevi, but was significantly greater than expression in conventional nevi and approached significance after multiple comparisons corrections for malignant melanoma. Thus, a high level of P21 expression makes a tumor more likely to be a typical or atypical Spitz nevus than a malignant melanoma, especially when coupled with a low Ki-67 index and weak expression of fatty acid synthase. These immunohistochemical observations support the concept that atypical Spitz nevi are distinct lesions of borderline biologic behavior residing between Spitz nevi and malignant melanoma. The study also compared a large array of histologic features of 16 cases of typical Spitz nevi in children with 12 typical Spitz nevi in adults. The adult lesions were significantly more likely to be intradermal and to display dermal fibroplasia, but were histologically similar to their pediatric counterparts in all other respects.
Apoptosis is important for maintenance of tissue homeostasis and often dysregulated in cutaneous neoplasms. The apoptosis inhibitor survivin is expressed in melanoma and non-melanoma skin cancers and benign keratinocytic lesions. Its expression has not been studied in melanocytic nevi.
We determined the expression pattern of survivin in benign melanocytic nevi in comparison to markers of proliferation and apoptosis.
Six cases of each of the following melanocytic nevi were retrieved from a dermatopathology archive: compound dysplastic nevus, intradermal nevus, compound nevus, neurotized intradermal nevus, and Spitz nevus. Survivin expression was evaluated by in situ hybridization. Apoptotic and proliferation indices were calculated by counting immunoreactive cells in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling and proliferating cell nuclear antigen immunostained sections, respectively.
All nevi, regardless of histologic type, expressed survivin. Compound melanocytic lesions expressed survivin in both epidermal and dermal compartments. The apoptotic rate was low for dysplastic, compound, and Spitz nevi, and apoptotic cells were not identified in any neurotized nevus. The proliferative index was highest for Spitz nevi, while all other nevi demonstrated rare positive cells.
Survivin is consistently expressed in benign melanocytic lesions, while apoptotic cells are rarely identified, suggesting the dysregulation of apoptotic pathways with the accumulation of cells in these neoplasms.
The clinical and histopathological similarities of nodular melanoma and Spitz nevus currently still make a definitive diagnosis difficult. We report here a case of nodular melanoma that was extremely difficult to diagnose both clinically and histopathologically. The primary tumor was a blackish nodule on the scalp and biopsy was performed for pathological diagnosis. Although our first impression was malignant melanoma, we asked two dermatopathologists for second opinions; however, one diagnosed a melanoma and the other a Spitz nevus. Faced with this clinical dilemma, an operation was performed with sentinel node biopsy. Only one sentinel node suggested a metastasis. Histopathological diagnosis to establish whether it was a melanoma metastasis or nodal nevi was also difficult, and we again asked for second opinions from another dermatopathologist in the USA. According to its clinical course and the histopathology of the sentinel lymph node with additional immunohistochemistry, this case was finally diagnosed as a nodular melanoma (T4aN1aM0, stage IIIA). To date, the patient has been given five courses of chemotherapy at 6-month intervals, with no local recurrence or distant metastases so far.
The last two decades have seen spectacular advances in our understanding of the biology of melanoma and, in particular, have elucidated the mechanisms operative in disease initiation and progression. With respect to the former, the genetics of melanoma and in particular the impact of genetic defects on dysregulation of the cell cycle are key issues in malignant transformation and are a major focus of this review. With respect to the latter, consideration also is given to the acquisition of growth factor autonomy and the capacity for invasion and metastasis from the standpoint of cell adhesion, motility, and matrix digestion. These events have specific morphologic correlates that will be briefly addressed. Where relevant, we will address certain of the modern pharmacogenetic strategies that flow from these novel observations concerning melanoma biology.
This study reports the identification of an autoantibody in the sera of some patients with systemic lupus erythematosus that reacted with nuclear antigen(s) of proliferating cells. The autoantibody was initially detected by the observation that it did not react in immunofluorescence with nuclei of renal tubular or glomerular cells, nor with hepatic parenchymal cells, but only reacted with scattered cells in the interstitial tissue of these two organs. In contrast, many lymphocytes in lymph node follicles, spleen, and thymus reacted with this antinuclear antibody. Normal peripheral blood lymphocytes did not show nuclear staining but after mitogenic stimulation, 20% of cells became positive. Nuclear staining was not restricted to lymphocytes but was also observed in several tissue culture cells lines such as Hep-2 cells (human epithelial carcinoma), Ehrlich ascites tumor cells, and baby hamster kidney cells. The reactive nuclear antigen(s) was soluble in physiologic saline and reacted with serum autoantibody to give a precipitin line in immunodiffusion that was immunologically distinct from DNA and other known nuclear antigen-antibody precipitating systems. Autoantibodies to proliferating cell nuclear antigen(s) might serve as useful biologic markers to study stimulated lymphocytes and other proliferating cells.
Nuclear patterns of cyclin (PCNA) distribution that subdivide S-phase (determined using PCNA autoantibodies specific for this protein) as well as [3H]thymidine incorporation followed by autoradiography have been used to determine the S-phase synchrony of homophasic polykaryons produced by polyethylene glycol (PEG)-induced fusion of populations of mitotic transformed human amnion cells (AMA) exhibiting the following average distribution of phases: prophase, 9%, metaphase, 60% (including early and late prometaphase), anaphase, 3.8%, telophase, 26.2% and interphase, 1%. Both synchronous and asynchronous polykaryons were generated from these fusions; the latter being frequently observed only amongst populations of multinucleated cells having three or more nuclei. These results are taken to imply that individual nuclei in these polykaryons can control cyclin distribution and DNA synthesis in spite of the fact that they share a common cytoplasm.
Pulse-chase experiments have revealed that cyclin, the auxiliary protein of DNA polymerase-delta, is stable during the transition from growth to quiescence in 3T3 cells. Immunoblotting together with immunofluorescence analysis has shown that the amount of cyclin after 24 h of quiescence is 30-40% of that of growing cells and that it presents a nucleoplasmic staining. Immunofluorescence studies show the existence of two populations of cyclin during the S phase, one that is nucleoplasmic as in quiescent cells and is easily extracted by detergent, and another that is associated to specific nuclear structures. By using antibromodeoxyuridine immunofluorescence to detect the sites of DNA synthesis, it was shown that the staining patterns of the replicon clusters and their order of appearance throughout the S phase are identical to those observed for cyclin. Two-dimensional gel analysis of Triton-extracted cells show that 20-30% of cyclin remains associated with the replicon clusters. This population of cyclin could not be released from the nucleus using high-salt extractions. This demonstrates that cyclin is tightly associated to the sites of DNA replication and that it must have a fundamental role in DNA synthesis in eukaryotic cells.
When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone. This result suggests that cytosol-depleted permeabilized DNA repair-defective human fibroblasts and HeLa DNA polymerase delta might be exploited to provide a functional assay for purifying active DNA repair factors from DNA repair-proficient cells without a preknowledge of their function.
The polypeptides synthesized during the cell cycle of HeLa cells were analyzed by means of two-dimensional gel electrophoresis followed by fluorography under conditions in which the position of 700 polypeptides (acidic and basic) could be reproducibly assessed. Mitotic cells obtained by mechanical detachment and synchronized cells in other stages of the cell cycle were labeled with [35S]methionine for 30-min pulses or for long terms starting at the beginning of each phase. Visual comparison of the polypeptide maps obtained in the different stages of the cell cycle showed that these were strikingly similar, and there was no indication that the synthesis of any of the detected polypeptides was confined to only one of the cell cycle phases. Quantitation of 99 abundant polypeptides (acidic and basic) in pulse-labeled and long-term labeled cells revealed that the relative amount (i.e., the rate of synthesis) of most polypeptides, including total actin, alpha-actinin, 6 abundant basic nonhistone proteins, and 13 major acidic proteins present in Triton cytoskeletons, remains constant throughout the cell cycle. Among the few variable polypeptides (markers), we have identified alpha- and beta-tubulin (increase in M), the subunit of the 100-A filament protein "fibroblast type" (decreases in M), and a 36,000 mol wt acidic cytoarchitectural protein that increases in S. A few other unidentified polypeptides have also been found to vary in M and in M and G2, but no marker was found in G1.
• By now it is well recognized that there is a benign melanocytic nevus, common in the young and common enough in adults, that has histological features that are confusable with those of malignant melanoma. The anomaly is usually referred to as benign juvenile melanoma, sometimes as Spitz's nevus, and, by some histopathologists, as spindle and epithelioid cell nevus. All the histological subtleties and variations of the condition are still not fully appreciated and some of them are still being misinterpreted as those of malignant melanoma.
We herewith present a study designed to clarify the issues and offer firm criteria for histological differentiation of the nevus in point from malignant melanoma.
We also suggest a new name for it and supporting arguments therefor.
(Arch Dermatol 114:1811-1823, 1978)
A 36K MW polypeptide was previously identified by two-dimensional gel electrophoresis whose relative proportion increases in S phase of HeLa cells, and following viral transformation of mouse 3T3B and hamster BHK21 cells. We report here experiments that show that this polypeptide (isoelectric focusing (IEF 49)) is localized in the nucleus and that its relative proportion decreases dramatically under conditions for which there is a decrease in the rate of cell proliferation, due for example, to the formation of giant HeLa cells by X-ray irradiation or the senescence of human skin fibroblasts. It is present in negligible amounts in non-cycling cells of adult tissues. These studies also revealed and unrelated cytoplasmic polypeptide (IEF 52; tropomyosin related; coordinates 35/1.43) whose relative proportion increased significantly and reproducibly with a decrease in the rate of cell proliferation. It is suggested that IEF 49 may be a useful marker for cycling cells.
Of 13 cases of juvenile melanoma in this series, only one (7.7 percent) has had a clinically malignant and fatal course despite the similarity of the juvenile lesions to the malignant melanoma of adults. Juvenile melanoma may be distinguished histologically from adult melanoma in about one half the cases by the presence of giant cells in the former, which seldom occur in the latter. There is a precipitous rise in the capacity of melanomas to metastasize after puberty despite the histologic similarity to the usually nonmetastasizing juvenile melanoma. The possible influence of sex-linked hormonal activation of the growth capacity of melanomas at the age of puberty seems a logical conclusion. Accordingly, since metastases from juvenile melanomas occur only rarely, conservative surgery, rather than the radical surgery usually indicated for adult melanomas, seems justified.
By now it is well recognized that there is a benign melanocytic nevus, common in the young and common enough in adults, that has histological features that are confusable with those of malignant melanoma. The anomaly is usually referred to as benign juvenile melanoma, sometimes as Spitz's nevus, and, by some histopathologists, as spindle and epithelioid cell nevus. All the histological subtleties and variations of the condition are still not fully appreciated and some of them are still being misinterpreted as those of malignant melanoma. We herewith present a study designed to clarify the issue and offer firm criteria for histological differentiation of the nevus in point from malignant melanoma. We also suggest a new name for it and supporting arguments therefor.
The expression of proliferating cell nuclear antigen (PCNA), also called cyclin, was quantified in the cell lines SP2/0 and MOLT-4 and in mouse splenocytes induced to proliferate in vitro with mitogens. Autoantibody from a patient with systemic lupus erythematosus was used to label PCNA in cell suspensions after the cells had been fixed and permeabilized. In the same cells DNA was stained by propidium iodide. The cells were then analysed by flow cytometry for PCNA and DNA content. The PCNA profiles in proliferating spleen cells and the cell lines were similar. Most G0-G1 cells did not express significant amount of PCNA. A dramatic increase in PCNA immunofluorescence was observed in late G1 cells, and further increases were observed in S-phase cells. G2-M cells showed a reduced level of PCNA immunofluorescence relative to S-phase cells but were still elevated relative to G0-G1 cells. Proliferating cells arrested at the G1-S boundary by exposure to cytosine arabinoside showed an increased PCNA immunofluorescence as compared to unstimulated cells.
The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.
Spitz nevi and malignant melanoma may be difficult to differentiate histologically. We applied an antibody to proliferating cell nuclear antigen (PCNA) to cases of Spitz nevi and malignant melanoma to determine if there were differences in reactivity. Cases were selected from the material of the Department of Pathology, University of Iowa Hospital. Formalin-fixed, paraffin-embedded cases were immunohistochemically stained with anti-PCNA (PC 10) with use of avidin-biotin complex technique. The cases were evaluated for the presence or absence of reactivity, staining intensity, and semiquantitative percentage of positive cells. Positive staining was observed in six of the 10 cases of Spitz nevi. The intensity of staining was weak and the number of positive cells varied from 10% to 40%. Positive staining was observed in all 10 cases of malignant melanoma. In eight of these 10 cases the intensity of staining was strong and the number of positive cells was greater than 40%. In the remaining two cases of melanoma, the intensity of staining was weak and the percentage of positive cells was 20-30%. These results indicate that PCNA staining may be observed in both Spitz nevi and malignant melanoma. Presence or absence of reactivity cannot be used in distinguishing these processes. However, a pattern of strong, diffuse reactivity may be used as an adjunct for supporting a diagnosis of melanoma.
Accepted for publication Dec Reprint requests: NealS.Penneys, MD, PhD, Division of Dermatology, 1402 S
- Louis Saint
- University
b Saint Louis University. Accepted for publication Dec. 30, 1994. Reprint requests: NealS.Penneys, MD, PhD, Division of Dermatology, 1402 S. Grand Blvd., St. Louis, MO 63104. Copyright @ 1995 by the American Academy of Dermatology, Inc.
Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: associate with DNA replication sites
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