Multiple lysosomal trafficking phenotypes in metastatic mouse mammary tumor cell lines.

Although altered synthesis and trafficking of lysosomal proteins and their receptors are associated with a wide range of human and rodent malignancies, the basis for their involvement remains obscure. Here we describe findings on a set of mouse mammary tumor cell lines that we are using as a model to study the role of these proteins in oncogenesis and tumor progression. Three distinct proteinase-secreting phenotypes were identified among the metastatic cell lines of the set. Two phenotypes displayed a high level of secretion of cathepsin L and the third was characterized by elevated secretion of matrix metalloproteinase 9 (MMP-9). The two cathepsin L-secreting phenotypes were distinct in that they displayed differences in cathepsin trafficking, expression of mannose 6-phosphate/insulin-like growth factor receptor and expression of proliferin, a mannose-phosphorylated angiogenic factor. Although cells representing all three phenotypes are capable of dissemination to distant organs when implanted into mouse mammary glands, only cells with the MMP-9 phenotype were found to be capable of direct intravasation. These findings indicate that multiple proteinase-secreting phenotypes can arise from the same tumor and suggest that cathepsin L and other lysosomal proteins may play a role in dissemination of tumor cells via the lymphatic system.

Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis.

Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells.

The MCF10 series of cell lines was derived from benign breast tissue from a woman with fibrocystic disease. The MCF10 human breast epithelial model system consists of mortal MCF10M and MCF10MS (mortal cells grown in serum-free and serum-containing media, respectively), immortalized but otherwise normal MCF10F and MCF10A lines (free-floating versus growth as attached cells), transformed MCF10AneoT cells transfected with T24 Ha-ras, and premalignant MCF10AT cells with potential for neoplastic progression. The MCF10AT, derived from xenograft-passaged MCF10-AneoT cells, generates carcinomas in approximately 25% of xenografts. We now report the derivation of fully malignant MCF10CA1 lines that complete the spectrum of progression from relatively normal breast epithelial cells to breast cancer cells capable of metastasis. MCF10CA1 lines display histologic variations ranging from undifferentiated carcinomas, sometimes with focal squamous differentiation, to well-differentiated adenocarcinomas. At least two metastasize to the lung following injection of cells into the tail vein; one line grows very rapidly in the lung, with animals moribund within 4 weeks, whereas the other requires 15 weeks to reach the same endpoint. In addition to variations in efficiency of tumor production, the MCF10CA1 lines show differences in morphology in culture, anchorage-independent growth, karyotype, and immunocytochemistry profiles. The MCF10 model provides a unique tool for the investigation of molecular changes during progression of human breast neoplasia and the generation of tumor heterogeneity on a common genetic background.

Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates: a novel two-dimensional liquid chromatography/mass spectrometry method.

A novel two-dimensional two-column liquid chromatography/mass spectrometry (LC/MS) technique is described in this work, where chromatofocusing (CF) has been coupled to nonporous reversed-phase (NPS-RP) HPLC to separate proteins from human breast epithelial whole cell lysates. The liquid fractions from NPS-RP-HPLC are readily amenable to direct on-line analysis using electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (ESI-TOFMS). A key advantage of this technique is that proteins can be 'peeled off' in the liquid phase from the CF column according to their isoelectric points (pI) in the first chromatographic separation dimension. The NPS-RP-HPLC column further separates these pI-focused fractions based upon protein hydrophobicity as the second chromatographic dimension. The third dimension involves on-line molecular weight determination using ESI-TOFMS. As a result, this method has the potential to be fully automated. In addition, a 2-D protein map of pI versus molecular weight is generated, which is analogous to a 2-D gel image. Thus, this technique may provide a means to study differential expression of proteins from whole cell lysates.

Tyrosine phosphorylation of an erbB-2 related p90 protein induced by estrogen in human breast epithelial cells.

Estrogen induced erbB-2 tyrosine kinase activity in human breast epithelial cells irrespective of estrogen receptor expression. MCF10A is an immortal normal human breast epithelial cell line which does not express estrogen receptor. After treatment of MCF10A cells with estradiol-17beta (E2), a phosphorylated 90 kDa protein which co-immunoprecipitates with p185erbB-2 is detected. The response is transient, detected after 1-5 min exposure to E2, and dose dependent, occurring at 10-10 M E2. A similar response was observed for MCF10A cells transfected with an estrogen receptor, estrogen receptor expressing MCF-7 cells, and estrogen receptor-negative MDA-MB-435 cells but at 10-11 M E2. Overexpression of c-erbB-2 in MCF10A cells prolonged the phosphorylated p90 response to E2.

Cyclin D1 overexpression in a model of human breast premalignancy: preferential stimulation of anchorage-independent but not anchorage-dependent growth is associated with increased cdk2 activity.

Cyclin D1 is frequently overexpressed in human breast ductal carcinoma in situ (DCIS) specimens, which confer a high risk for the development of infiltrating ductal carcinoma. If causally involved in the genesis of human breast malignancy, cyclin D1 may represent an interesting target for chemopreventive approaches, as it sits at the junction of many growth factor and hormonal pathways. We have used the MCF-10A human breast cell line, derived from a mastectomy containing a low risk premalignant lesion, as a model system. Three cyclin D1 transfectants exhibited physiologically relevant levels of transgene overexpression, but no coordinate overexpression of other cell cycle related genes. Proliferation assays, flow cytometry, and cdk enzymatic assays of anchorage-dependent proliferation indicated only a minimal and transient effect of cyclin D1. In contrast, cyclin D1 overexpression significantly stimulated anchorage-independent colonization in soft agar or methylcellulose, accompanied by greater Gl-S progression. The cdk4 activity of the control- and cyclin D1 transfectants in colonization assays was comparable, but the cdk2 activity was higher in the latter. Injection of control- and cyclin D1 transfected MCF-10A cells in matrigel into nude mice failed to produce tumors within 1.5 years. The data suggest that cyclin D1 overexpression is an early feature of breast neoplastic progression, and can contribute to cancer development through the promotion of colonization.

Progression of premalignant MCF10AT generates heterogeneous malignant variants with characteristic histologic types and immunohistochemical markers.

The MCF10AT premalignant human breast epithelial cells form benign ductal structures in immunodeficient mice which sporadically progress to carcinoma in situ and invasive cancers of different histologic types. MCF10CA1 cell lines are malignant variants derived by serially passing small pieces of tumors in athymic mice before establishing cells in culture. As these MCF10CA1 variants gave rise to heterogeneous tumors, some cell lines were cloned. Inoculated into immunodeficient mice, these variants produce squamous carcinomas with an undifferentiated component or adenocarcinomas also with an undifferentiated component. Immunohistochemistry utilized antibodies against DF3, c-erbB-2, cyclin Dl, m keratin, p keratin, p53, B72.3 and estrogen receptor. We detected characteristic patterns for squamous carcinomas, for adenocarcinomas, and for each undifferentiated component, that is the undifferentiated components of the squamous and glandular carcinomas were distinct. Only adenocarcinomas were focally ER positive. One uncloned variant that produced cancers with a glandular component, MCF10CA1h, was cloned and cells were injected into mice. This clone produced only undifferentiated carcinomas that, compared to tumors formed by the parental uncloned variant, had lost ER, DF3 and c-erbB-2 expression, but more strongly expressed p53. Our data demonstrate the potential of the premalignant MCF10AT model to generate heterogeneity, including both estrogen receptor-positive as well as estrogen receptor-negative tumors, during progression.

Nontransgenic models of breast cancer.

Numerous models have been developed to address key elements in the biology of breast cancer development and progression. No model is ideal, but the most useful are those that reflect the natural history and histopathology of human disease, and allow for basic investigations into underlying cellular and molecular mechanisms. We describe two types of models: those that are directed toward early events in breast cancer development (hyperplastic alveolar nodules [HAN] murine model, MCF10AT human xenograft model); and those that seek to reflect the spectrum of metastatic disease (murine sister cell lines 67, 168, 4T07, 4T1). Collectively, these models provide cell lines that represent all of the sequential stages of progression in breast disease, which can be modified to test the effect of genetic changes.

Xenograft models of premalignant breast disease.

Dysplastic and hyperplastic proliferative lesions with graded severity of atypia are recognized in a number of tissues and are generally suspected to be premalignant, that is to say at high risk for further progressing to carcinoma in situ and invasive cancer. However, few xenograft models of premalignancy for any organ site have been successfully developed. A good model of human premalignant breast disease would lead to lesions which resemble high risk human breast disease in xenografts and sporadically progress to invasive cancer with time. In this chapter the use of breast tissue pieces and epithelial cells for establishment of xenografts and the development of human breast epithelial cell lines that form premalignant xenograft lesions are described. MCF10AT cells not only form simple differentiated ducts which persist in xenografts and sporadically progress to carcinoma, but also form intermediate proliferative lesions resembling proliferative disease without atypia, atypical hyperplasia, and carcinoma in situ.

Rapid screening of protein profiles of human breast cancer cell lines using non-porous reversed-phase high performance liquid chromatography separation with matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis.

Non-porous reversed-phase (NP-RP) HPLC has been used to rapidly generate protein profiles of whole cell lysates of human breast cancer cell lines. The non-porous packing material used was silica coated with C18, which provided rapid separation with high collection efficiency of proteins from cell lysates. This method was used to study the differences in protein profiles among normal cells and fully malignant cells that share a common genetic background. The highly expressed proteins in each cell type were separated and collected in the liquid state where they were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to obtain the molecular weight of the proteins. The protein fractions were then subjected to tryptic digestion and analyzed by pulsed delay extraction (PDE)-MALDI-TOFMS to obtain the peptide maps. The expressed proteins were identified based upon the molecular weight and peptide map using database-searching procedures. It is shown that key cancer-related proteins can be detected and identified which may be potentially used as biomarkers for cancer detection.

A novel orthotopic model of breast cancer metastasis to bone.

Breast cancer affects approximately one woman in twelve and kills more women than any other cancer. If detected early, patients have a five year survival rate of 66%, but once metastatic disease has developed, there is no effective treatment. About 70% of patients with metastatic disease have bone involvement, while lungs and liver are the other common targets. Bone metastases cause severe pain, pathological fractures and hypercalcaemia and thus are a significant clinical problem. The development of new therapies for metastatic breast carcinoma depends on a better understanding of the mechanism of homing of the tumour cells to bone, liver and lungs and the factors required for their growth in these organs. Research on mechanisms of breast cancer metastasis, particularly to bone, has relied on in vitro studies or on tumour models in which the inoculation route is designed to promote delivery of tumour cells to a specific organ. Metastases in bone are achieved by inoculation into the right ventricle of the heart. To our knowledge there has been no report of a model of metastatic spread from the mammary gland to distant sites which reliably includes bone. In this paper, we describe our recent development of a novel murine model of metastatic breast carcinoma. The new model is unique in that the pattern of metastatic spread closely resembles that observed in human breast cancer. In particular, these murine breast tumours metastasise to bone from the primary breast site and cause hypercalcaemia, characteristics not normally found in murine tumours, but common in human disease. Furthermore, in a preliminary characterisation of this model, we show that secretion of parathyroid hormone-related protein, a role for which has been implicated in breast cancer spread to bone, correlates with metastasis to bone. This model therefore provides an excellent experimental system in which to investigate the factors that control metastatic spread of breast cancer to specific sites, particularly bone. The special advantage of this system is that it involves the whole metastasis process, beginning from the primary site. Existing models consider mechanisms that pertain to growth of tumour once the site has been reached. An understanding of the regulation of these factors by potential therapeutic agents could lead to improvement in therapies designed to combat metastatic disease. For the first time, this development will allow exploration of the molecular basis of site-specific metastasis of breast cancer to bone in a clinically relevant model.

Differential display, subtractive hybridization, and application of methodology to search for point mutations to identify genetic defects responsible for progression in MCF10AT model of human breast disease.

We describe initial studies utilizing three methods to detect differences in gene expression: (i) differential display with polyT-anchored primers; (ii) differential display with RNA arbitrarily primed polymerase chain reaction (RAP-PCR), and (iii) cDNA subtractive hybridization. Subtractive hybridization, which detects qualitative differences in gene expression, revealed no differences between a human cell line (MCF10A), originated by spontaneous immortalization of breast epithelial cells, and MCF10CA1 cells, a recently derived fully malignant, metastatic variant. The standard method of differential display with polyT anchored primers detected in excess of 100 differentially displayed bands but differential expression could seldom be verified by Northern blotting. However, RAP-PCR products frequently represent the coding region and fewer bands are detected. One gene of interest is a zinc finger protein which may be expressed more in the preneoplastic lesion-forming cells than in nonlesion-forming cells. Because most bands are not consistently differentially displayed among the variants of the MCF10 model, we suspect that point mutations rather than differential quantitative gene expression is responsible for some stage of progression. We propose that differential display of RAP-PCR products on nondenaturing gels might also detect point mutation differences.

Surgical management of thyroid and parathyroid disorders.

This article begins with a discussion of thyroid anatomy and physiology and then proceeds with an in-depth study of the area of focus, thyroid and parathyroid disorders, including thyroid nodules and cancer, multinodular goiter, and hyperparathyroidism. Complications of thyroid and parathyroid surgery are discussed.

Plasmacytomas of the head and neck.

Plasmacytomas are rare tumors that often appear in the head and neck region and are characterized by a monoclonal proliferation of plasma cells. On both clinical presentation and pathologic examination these tumors may be confused with more common tumors of the head and neck. The purpose of this article is to review our experience with these rare neoplasms, with emphasis on clinical, pathologic, and therapeutic features. On retrospective chart review, we identified 20 patients with the diagnosis of plasmacytoma of the head and neck region at the Cleveland Clinic Foundation between 1976 and 1993. Records were reviewed with regard to initial symptoms, location of the neoplasm, diagnostic evaluation, treatment modalities, and survival. Of the 20 cases we identified, the tumor arose in the sinonasal/nasopharyngeal region in 11 (55%). Two cases (10%) represented medullary plasmacytomas, arising in the clavicle and presenting as supraclavicular masses. The mean follow-up was 60.2 months (range 6 to 131 months). In 15 of the 20 cases, immunohistochemistry staining for immunoglobulin light chain production was conducted. One of the two cases (50%) classified as medullary plasmacytoma demonstrated conversion to multiple myeloma, whereas only 2 of 18 cases of extramedullary plasmacytoma (11%) converted to multiple myeloma. The primary modality of treatment was radiation therapy with typical doses of 4500 to 6000 cGy. Kaplan-Meier survival estimates demonstrated 95% survival at 1 year, 82% survival at 5 years, and 10-year estimated survival of 72%. Plasmacytomas of the head and neck region are rare and on initial evaluation must be distinguished from multiple myeloma. The diagnostic evaluation includes appropriate radiologic and pathologic studies including immunohistochemistry. Despite the typical presentation as a locally destructive tumor, plasmacytomas are highly radiosensitive, and 70% to 80% survival may be obtained with the use of radiotherapy. Patients with plasmacytomas require long-term follow-up to detect conversion to multiple myeloma.

Transcriptional activation of functional endogenous estrogen receptor gene expression in MCF10AT cells: a model for early breast cancer.

Utilizing the MCF10AT xenograft model for progression of human proliferative breast disease, we detected expression of the endogenous estrogen receptor (ER) gene only in MCF10AneoT and cells of the MCF10AT system, all of which stably express a transfected mutated T24 Ha-ras gene. ER transcripts were undetectable in the parental MCF10A cells and in MCF10A cells transfected with normal c-Ha-ras or vector. ER transcripts expressed in MCF10AT cells contain a normal full-length ER coding region and direct synthesis of a normally sized ER protein. The protein is functional based on its ability to mediate estradiol (E2)-induced increases of transcription from both endogenous and exogenous E2-regulated genes. Transcriptional activation of the endogenous ER gene does not appear to be related to a change in methylation status of the gene since a diagnostic CpG site in exon 1 that is methylated in ER-negative breast tumors and completely unmethylated in ER-positive breast tumors is hypomethylated to the same extent in ER-negative MCF10A cells and ER-positive MCF10AT cells. E2 increased both the number and size of soft-agar colonies formed by MCF10AT3c cells, a line from a third generation MCF10AT xenograft lesion. This suggests that xenograft passage has selected for growth regulatory pathways that are E2-responsive and that identification of these pathways and their role in progression will aid in determining how E2 acts to increase risk of breast cancer.

Vagal paraganglioma: a review of 46 patients treated during a 20-year period.

BACKGROUND: Vagal paragangliomas (VPs) arise from paraganglia associated with the vagus nerve. Approximately 200 cases have been reported in the medical literature. Because of their rarity, most information regarding these tumors has arisen from case reports and small clinical series. OBJECTIVE: To detail the clinicopathologic features of 46 patients with VP with an emphasis on the role of a multidisciplinary skull base team in both the successful extirpation and rehabilitation. DESIGN: Retrospective review of 46 patients with VP managed by a single skull base team. SETTING: An academic tertiary medical center. RESULTS: Forty-six patients were treated over a 20-year period (1978-1998). Ten (22%) demonstrated intracranial extension. There was a history of familial paragangliomas in 9 (20%) of the patients. The incidence of multicentric paragangliomas was 78% in patients with familial paragangliomas vs 23% in patients with nonfamilial paragangliomas. Management of this group of 46 patients consisted of surgery (n = 40), radiation therapy (n = 4), and observation (n = 2). The operative approach consisted of a transcervical excision often combined with a transtemporal or lateral skull base approach as dictated by the tumor extent. Postoperative cranial nerve deficits were common, and, as such, aggressive rehabilitation was a vital component in the management of these tumors. CONCLUSIONS: The management of VP and its associated cranial nerve deficits remains a difficult clinical problem. Options for treatment include surgical resection, radiation therapy, and, in selected cases, observation. Surgical extirpation requires a multidisciplinary skull base team to achieve complete tumor resection. Radiation therapy is reserved for elderly patients and patients at risk for bilateral cranial nerve deficits. Rehabilitation of cranial nerve deficits is an integral part of the management of VP.

Altered expression of c-erbB-2, DF3, b72.3, p53 and Ki-67 with progression and differentiation to two distinct histologic types of invasive carcinoma in the MCF10AT human xenograft model of proliferative breast disease.

Human breast epithelial MCF10AT cells form simple ducts in nude/beige mice which eventually become hyperplastic and sporadically progress to carcinomas. Altered immunohistochemical detection of c-erbB-2, DF3, B72.3, p53 and Ki-67 was observed with progression and differentiation to two distinct histologic types of invasive carcinoma. c-erbB-2 and DF3 were detected in 50% and 18% of lesions at the stage of atypical hyperplasia and expression increased to 78% and 54% in invasive adenocarcinomas. In contrast, a group of six unusual undifferentiated tumors with squamoid features did not express c-erbB-2 or DF3, but both B72.3 (4/6) and p53 (6/6) were detected.

Use of tumor lines with selectable markers in assessing the effect on experimental metastases of combination chemotherapy with alkylating agents.

High-dose chemotherapy with a 3-day regimen of cyclophosphamide, cisplatin, and carmustine was used to treat mice bearing experimental lung metastases of mammary tumor cell lines with selectable markers (lines 66cl4 and 4TO7). Cloning of lung cells at various times after treatment revealed a rapid 3 to 4 log loss of clonogenic tumor cells, down to undetectable levels. However, after several weeks, clonogenic tumor cells reappeared in the lungs; few cures were obtained even when mice had a relatively low tumor burden when treated with chemotherapy. Splenocyte numbers and response to Concanavalin A indicated a transient immunosuppression. In one experiment, mice were treated with a second round of chemotherapy 3 weeks after the first. The number of clonogenic cells per lung again dropped, but regrowth of cells was rapid, and no cures were obtained. Inoculation of tumor-bearing mice s.c. after chemotherapy with lethally irradiated cells of the highly immunogenic tumor cell line 4TO7-IL-2 had little effect on the rate of reappearance of line 4TO7 in lungs, but subsequent growth of tumor cells in lungs was slowed. This model system can be used to test the efficacy of additional immunotherapy and chemotherapy regimens on minimal residual metastatic disease after high-dose chemotherapy, when remaining metastatic cells are apparently dormant.