Loss of adipose tissue and plasma phospholipids: relationship to survival in advanced cancer patients.

BACKGROUND & AIMS: Extensive loss of adipose tissue is a key feature of cancer cachexia. Advanced cancer patients also exhibit low plasma phospholipids. It is not known whether these processes coincide across the cancer trajectory nor has their relationship with survival been defined. Changes in adipose tissue mass and plasma phospholipids were characterized within 500days prior to death and prognostic significance assessed. METHODS: Adipose tissue rate of change was determined in a retrospective cohort of patients who died of colorectal and lung cancers (n=108) and who underwent >2 computed tomography scans in the last 500days of life. Plasma phospholipid fatty acids were measured prospectively in a similar cohort of patients with metastatic cancer (n=72). RESULTS: Accelerated loss of adipose tissue begins at 7months from death reaching an average loss of 29% of total AT 2months from death. Plasma phospholipid fatty acids were 35% lower in patients closest to death versus those surviving >8months. Losses of phospholipid fatty acids and adipose tissue occur in tandem and are predictive of survival. CONCLUSIONS: Depletion of plasma phospholipids likely indicates a deficit of essential fatty acids in the periphery which may contribute to loss of adipose tissue.

Synthesis of specific fatty acids contributes to VLDL-triacylglycerol composition in humans with and without type 2 diabetes.

AIMS/HYPOTHESIS: It is recommended that patients with diabetes reduce their intake of saturated fat and increase their intake of monounsaturated fat or carbohydrate. However, high-carbohydrate diets may result in higher saturated fatty acids in VLDL-triacylglycerol. This is attributed to de novo lipogenesis, although synthesis of specific fatty acids is rarely measured. The objective of this study was to examine the contribution of de novo fatty acid synthesis to VLDL-triacylglycerol composition. It was hypothesised that levels of total and de novo synthesised fatty acids would increase with increased carbohydrate intake in diabetic participants. METHODS: Seven individuals with type 2 diabetes mellitus and seven matched non-diabetic controls consumed two diets differing in fat energy (lower fat <25%, higher fat >35%) for 3 days in a randomised crossover design. Blood samples were drawn before and 24 h after the ingestion of (2)H-labelled water. RESULTS: In the control participants, the higher-fat diet resulted in a 40% reduction in VLDL-triacylglycerol fatty acids because of decreases in myristic, palmitic, palmitoleic and linoleic acids, but the opposite trend occurred in participants with diabetes. The lower-fat diet increased the fractional synthesis rate by 35% and 25% in the control and diabetes participants, respectively (range: 0-33%). Palmitate accounted for 71% of fatty acids synthesised (range: 44-84% total de novo synthesised fatty acids). CONCLUSIONS/INTERPRETATION: (2)H incorporation was used for the first time in humans showing variability in the synthesis rate of specific fatty acids, even palmitic acid. A lower-fat diet stimulated saturated fatty acid synthesis at high rates, but no net stimulation of synthesis of any fatty acid occurred in the diabetes group. The implications of this finding for our understanding of lipid metabolism in diabetes require further investigation.

Scalp metastases mimicking alopecia areata. First case report of placental site trophoblastic tumor presenting as cutaneous metastasis.

BACKGROUND: Placental site trophoblastic tumor (PSTT) is a rare neoplasm of intermediate trophoblastic cells of the placenta. There is a wide range of biologic behavior, with metastases occurring in about 15% of cases. Cases with metastases have all presented with abnormal vaginal bleeding or gynecological symptoms. METHODS: We describe a 31-year-old G3P3003 woman who presented with several alopecic patches resembling alopecia areata, which on biopsy proved to be metastatic, previously unsuspected, PSTT. CONCLUSIONS: This represents the first case in which PSTT presented initially with metastases, and specifically, with cutaneous metastases. A new primary tumor source of alopecia neoplastica is presented. The role of mitotic counts in predicting prognosis in PSTT is supported. Alopecia areata was mimicked very closely. Dermatologists should be alert to any features atypical of alopecia areata, including erythema, induration, or palpability, and maintain a low threshold for biopsy in atypical cases.

Expression of alpha 6 and beta 4 integrins in serous ovarian carcinoma correlates with expression of the basement membrane protein laminin.

The surface of a normal ovary is covered by a monolayer of epithelial cells that rest on a basement membrane. The glycoprotein laminin is the major noncollagenous protein present in the basement membrane. The integrins alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4 serve as cell surface receptors for laminin. During the progression of serous ovarian carcinoma, tumor cells are frequently exfoliated from the surface of the ovary, thereby losing contact with the basement membrane. This study was designed to determine whether alterations in integrin expression may be associated with the malignant phenotype of the primary ovarian tumor and exfoliated ovarian carcinoma cells in the ascites fluid. By immunohistochemical staining, the entire surface of epithelial cells of normal ovaries stained positively for beta 1, alpha 2, and alpha 3 integrins, whereas only the basal surface of the epithelial cells, where they are in contact with laminin, stained positively for alpha 6 and beta 4. The entire surface of epithelial cells of solid tumors from patients with serous ovarian carcinoma stained positively for beta 1, alpha 2, and alpha 3 integrins. In most cases, no intact basement membrane surrounded the tumor nests, and staining for alpha 6 and beta 4 was irregular. When present, the basement membrane stained positively for laminin, and the basal surface of the epithelial cells stained positively for alpha 6 and beta 4. Ovarian carcinoma ascites cells exhibited a distinct phenotype, with a significant decrease in expression of the alpha 6 and beta 4 integrin subunits. As alpha 6 and beta 4 integrin subunits are present at the basal surface of many epithelial cells and serve as receptors for laminin, it is possible that ovarian carcinoma epithelial cells may be released from the basement membrane of the ovary due to their deficit of alpha 6 and beta 4 integrin subunits.

Temperature-modulated pressure ulcers: a porcine model.

OBJECTIVE: A reliable porcine model was developed to facilitate investigations of pressure ulcer formation, healing, and prevention. In the present study, it was specifically used to study the relationship between applied temperature, applied pressure, and time of application in the formation of cutaneous and deep tissue injuries. DESIGN: An apparatus and procedure were created to simultaneously apply 12 metal discs (each with a diameter of 51mm) on the dorsal aspect of the swine, all at an equal pressure of 100mmHg, for a 5-hour period, while servo-controlling disc temperatures at either 25, 35, 40, or 45 degrees C. RESULTS: The severity of the resultant tissue injuries correlated with an increase in applied temperature. No damage was observed in the superficial or deep tissues underlying the sites of the 25 degrees C pressure applicators. In general, only deep tissue damage resulted from the application of a 35 degrees C temperature, whereas the application of higher temperatures caused both cutaneous and subdermal damage (the extent of necrosis being greater at the 45 degrees C sites). There was a high degree of reproducibility of these results among a large population of sites per temperature (n = 64) and number of animals investigated (n = 16). Furthermore, subsequent healing (monitored up to 4 weeks) was uniform for the degree of induced damage. Insights into pressure ulcer formation were also sought via systematic examination of histological sides and postmortem visual assessment over the 4-week period. CONCLUSION: It was concluded that this animal model of temperature-modulated pressure ulcers has the potential for significant use in all major areas of this field, ie, wound formation, healing, and prevention.

Human keratinocyte migration on type IV collagen. Roles of heparin-binding site and alpha 2 beta 1 integrin.

BACKGROUND: The migration of human keratinocytes is an early and important event in the re-epithelialization of cutaneous wounds. Type IV collagen, a ubiquitous basement membrane component, promotes keratinocyte migration. EXPERIMENTAL DESIGN: In this study, we sought to identify specific sites within the type IV collagen molecule that induce keratinocyte locomotion and to characterize the cell surface receptors involved. We first examined purified fragments of the type IV collagen molecule as substrates for keratinocytes in a phagokinetic migration assay. We then tested several synthetic peptides derived from the triple-helical region of type IV collagen, as well as antibodies against specific integrin subunits, for their ability to either support or inhibit keratinocyte migration on matrices of both type IV collagen and relevant peptide derivatives. RESULTS: Keratinocytes migrated on the triple-helical fragment to the same extent as they did on the native type IV collagen. The amino-terminal 7S and the carboxy-terminal NC1 regions of type IV collagen failed to support keratinocyte migration. In addition, Hep III peptide was active both in inhibiting keratinocyte migration on type IV collagen and in serving as a substrate matrix for migration. Peptide containing the amino acid sequence RGD did not influence cell migration on type IV collagen. A specific monoclonal antibody against the alpha 2 beta 1 integrin receptor significantly inhibited keratinocyte migration on matrices of both type IV collagen and Hep III peptide. CONCLUSIONS: Keratinocyte migration on type IV collagen involves the interaction of the alpha 2 beta 1 receptor with the Hep III region of the type IV collagen molecule.

Human keratinocytes adhere to and spread on synthetic peptide FN-C/H-V derived from fibronectin.

In a previous study, we reported that two synthetic peptides derived from the 33-kD carboxyl terminal cell/heparin-binding fragment of fibronectin A chain promoted keratinocyte adhesion but not spreading. Because keratinocytes are capable of spreading on the 33/66-kD fragments, we focused on identifying additional chemically synthesized peptides from the cell/heparin-binding fragments of fibronectin that might promote cell spreading. When plastic substrata were coated with peptide FN-C/H-V (WQPPRARI), which is derived from the carboxyl-terminal heparin-binding domain of all plasma fibronectin isoforms, keratinocytes adhered and displayed a spread morphology. In solution, soluble peptide FN-C/H-V inhibited cell spreading on intact fibronectin and on the 33/66-kD fragments. Furthermore, polyclonal antibodies raised against peptide FN-C/H-V also inhibited keratinocyte spreading on fibronectin and the 33/66-kD fragments. These data support the hypothesis that keratinocyte cell adhesion and cell spreading on fibronectin are mediated by multiple distinct domains and different regulatory processes.

Epiligrin, a component of epithelial basement membranes, is an adhesive ligand for alpha 3 beta 1 positive T lymphocytes.

The cutaneous T cell lymphomas (CTCL), typified by mycosis fungoides, and several chronic T cell mediated dermatoses are characterized by the migration of T lymphocytes into the epidermis (epidermotropism). Alternatively, other types of cutaneous inflammation (malignant cutaneous B cell lymphoma, CBCL, or lymphocytoma cutis, non-malignant T or B cell type) do not show evidence of epidermotropism. This suggests that certain T lymphocyte subpopulations are able to interact with and penetrate the epidermal basement membrane. We show here that T lymphocytes derived from patients with CTCL (HUT 78 or HUT 102 cells), adhere to the detergent-insoluble extracellular matrix prepared from cultured basal keratinocytes (HFK ECM). HUT cell adhesion to HFK ECM was inhibitable with monoclonal antibodies (mAbs) directed to the alpha 3 (P1B5) or beta 1 (P4C10) integrin receptors, and could be up-regulated by an activating anti-beta 1 mAb (P4G11). An inhibitory mAb, P3H9-2, raised against keratinocytes identified epiligrin as the ligand for alpha 3 beta 1 positive T cells in HFK ECM. Interestingly, two lymphocyte populations could be clearly distinguished relative to expression of alpha 3 beta 1 by flow cytometry analysis. Lymphokine activated killer cells, alloreactive cytotoxic T cells and T cells derived from patients with CTCL expressed high levels of alpha 3 beta 1 (alpha 3 beta 1high). Non-adherent peripheral blood mononuclear cells, acute T or B lymphocytic leukemias, or non-cutaneous T or B lymphocyte cell lines expressed low levels of alpha 3 beta 1 (alpha 3 beta 1low). Resting PBL or alpha 3 beta 1low T or B cell lines did not adhere to HFK ECM or purified epiligrin. However, adhesion to epiligrin could be up-regulated by mAbs which activate the beta 1 subunit indicating that alpha 3 beta 1 activity is a function of expression and affinity. In skin derived from patients with graft-vs.-host (GVH) disease, experimentally induced delayed hypersensitivity reactions, and CTCL, the infiltrating T cells could be stained with mAbs to alpha 3 or beta 1 and were localized in close proximity to the epiligrin-containing basement membrane. Infiltrating lymphocytes in malignant cutaneous B disease (CBCL) did not express alpha 3 beta 1 by immunohistochemical techniques and did not associate with the epidermal basement membrane. The present findings clearly define a function for alpha 3 beta 1 in T cells and strongly suggest that alpha 3 beta 1 interaction with epiligrin may be involved in the pathogenesis of cutaneous inflammation.

Human keratinocytes adhere to two distinct heparin-binding synthetic peptides derived from fibronectin.

Fibronectin is present at the dermal-epidermal junction in normal skin and is increased in skin tissues in inflammatory diseases, skin cancers, and wound repair. The present studies focused on further characterizing the interaction between fibronectin and keratinocytes, specifically addressing whether human keratinocytes utilize multiple adhesion promoting sequences within fibronectin. Initially, direct cell-binding assays were utilized in which keratinocyte adhesion to plastic substrata coated with fibronectin or proteolytic fragments of fibronectin was quantified. Intact fibronectin, a 75-kD proteolytic fragment containing the RGD sequence, and 33/66-kD cell adhesion/heparin binding fragments lacking the RGD sequence derived from the A and B chains of fibronectin, all promoted keratinocyte adhesion in a concentration-dependent manner. To further define putative cell-binding domains within the 33/66-kD fibronectin fragments, we studied three chemically synthesized peptides derived from the amino acid sequence of the 33-kD fragment of the fibronectin A chain: FN-C/H-I (YEKPGSPPREVVPRPRPGV), FN-C/H-II (KNNQKSEPLIGRKKT), and CS1 (DELPQLVTLPHPNLHGPEILDVPST). Substrata coated with either FN-C/H-I or FN-C/H-II promoted keratinocyte adhesion in a concentration-dependent and saturable manner, whereas peptide CS1 promoted no significant keratinocyte adhesion. In solution, both exogenous FN-C/H-I and FN-C/H-II partially inhibited keratinocyte adhesion to the 33/66-kD fibronectin fragments. Furthermore, antibodies prepared against these peptides also inhibited keratinocyte adhesion to the 33/66-kD fibronectin fragments. These data indicate that keratinocyte adhesion to fibronectin is mediated by multiple distinct amino acid sequences, at least two of which are localized to the carboxy-terminal heparin binding domain of fibronectin.

Human keratinocytes adhere to multiple distinct peptide sequences of laminin.

In normal human skin, basal layer keratinocytes of the epidermis are intimately associated with the lamina lucida of the basement membrane. Laminin, which is an 850-kD glycoprotein that has a cruciform shape by rotary shadowing and electron microscopy, is localized to the lamina lucida. The present study was aimed at further characterizing the interaction between laminin and cultured human keratinocytes. Initial studies revealed that laminin-coated substrata significantly promoted keratinocyte attachment in a concentration-dependent manner. To further define keratinocyte binding regions within laminin, a 440-kD proteolytic fragment of laminin was generated by limited chymotrypsin digestion, which renders laminin devoid of all terminal globular domains. Substrata coated with this 440-kD laminin fragment did not promote keratinocyte adhesion, suggesting that the globular domains may play an important role in cell adhesion. Based on these experiments, a series of chemically synthesized peptides derived from the A or B1 chains of laminin were studied. Among these, three peptides were found to be active in directly promoting keratinocyte adhesion: peptide F-9 (RYVVLPRPVCFEK) from the inner globule of the human B1 chain, TG-1 (RPVRHAQCRVCDGNSTNPRERH) from the top globule of the amino terminus (short arm) of the A chain, and GD-6 (KQNCLSSRASFRGCVRNLRLSR) from the large carboxy terminal globule at the end of the long arm of the A chain. In competition assays, these peptides in solution were shown to inhibit laminin-mediated keratinocyte adhesion. These studies show that normal human keratinocytes bind directly to laminin at a minimum of three distinct sites.

Tumor cell adhesive mechanisms and their relationship to metastasis.

Despite recent advances in the diagnosis and therapy of many forms of cancer, metastasis remains the major cause of death in cancer patients. As tumors progress they become increasingly heterogeneous, giving rise to aggressive subpopulations of tumor cells that subsequently invade local tissues, the lymphatics, and the circulatory system. This invasive behavior can ultimately lead to the widespread dissemination and metastasis of the primary tumor. In hematogenous metastasis, emboli consisting of tumor cells, host cells, platelets, and fibrin are transported to distant sites where they arrest in the microvasculature prior to extravasation. It is well accepted that tumor cell adhesion plays a fundamental role in many of the stages of the metastatic process. Tumor cell interactions with extracellular matrix components of tissue, tissue boundaries (basement membranes), or cell adhesion-promoting components of plasma; influence tumor cell motility, invasiveness, and many other important aspects of the metastatic tumor cell phenotype. Tumor cell adhesion also has a rate-limiting influence at various stages within the metastatic process, such as tumor cell arrest and extravasation. In addition, the ability of the immune system to recognize and successfully eradicated tumors is also highly dependent on the adhesion of activated lymphocytes to target tumor cells. Despite the rapid accumulation of information on the molecular basis of cell adhesion, our understanding of the relationship between tumor cell adhesion and hematogenous metastasis per se is fragmented and incomplete. Nevertheless, clear progress has been made, both in understanding the molecular basis of tumor cell adhesion and its relationship to the biology of tumor metastasis. New and exciting directions have been identified that are likely to yield direct benefits in developing new therapeutic or diagnostic approaches for malignant neoplasms. Our purpose is to briefly review the molecular basis of tumor cell adhesion from the standpoint of many of the receptors involved as well as their putative ligands. The relationship between specific tumor cell adhesion events and the formation of metastatic lesions is also addressed.

Human keratinocytes adhere to a unique heparin-binding peptide sequence within the triple helical region of type IV collagen.

The present studies were aimed at further characterizing the interaction between basement membrane molecules and normal cultured human keratinocytes because of the intimate association between basal keratinocytes and the basement membrane. The studies show that keratinocytes adhere to type IV collagen-coated substrata to a greater degree than substrata coated with similar concentrations of fibronectin and laminin. To further define cell-binding regions within type IV collagen, studies were performed using purified pepsin-generated triple helical fragments of type IV collagen and show that keratinocytes bind to sites within the triple-helical region of type IV collagen. To delineate specific cell adhesion promoting sequences, we studied a series of chemically synthesized peptides derived from the triple-helical region of type IV collagen. One peptide, designated Hep III, which is thirteen amino acids in length and binds heparin, was active in directly promoting keratinocyte adhesion. Furthermore, in competition assays, this peptide in solution was shown to inhibit keratinocyte adhesion to substrata coated with Hep III or intact type IV collagen. These studies show that keratinocytes bind directly to type IV collagen and chemically define a major cell-adhesion-promoting site within the triple helical region.

Human squamous carcinoma cells express complex defects in the control of proliferation and differentiation.

Four human squamous carcinoma cell (SCC) lines (SCC-9, SCC-13, SCC-15, and SCC-25) were studied to characterize their relative ability to control proliferation and differentiation. These experiments were based on previous data that established that in normal human keratinocytes three distinct and sequential steps are involved in the integrated control of proliferation and differentiation: 1) reversible growth-arrest at a predifferentiation state, 2) irreversible loss of proliferative potential, and 3) terminal differentiation. The current results show that SCC can show changes in the culture conditions required to undergo reversible growth-arrest and SCC can express partial or complete defects in their ability to irreversibly growth-arrest or terminally differentiate. For example, SCC-9 and SCC-25 cannot irreversibly growth-arrest or terminally differentiate, SCC-13 can irreversibly growth-arrest but cannot terminally differentiate, and SCC-15 can irreversibly growth-arrest and terminally differentiate to a moderate extent. These results therefore extend previous data by establishing that the malignant transformation of human epithelial cells does not simply result from defects in the control of terminal differentiation but rather from a combination of complex defects in the regulation of proliferation and differentiation.

Ability of normal human keratinocytes that grow in culture in serum-free medium to be derived from suprabasal cells.

The current studies were performed to determine from which regions of the skin keratinocytes that grow in vitro are derived. Normal human foreskin specimens were first separated by differential trypsinization into two suprabasal fractions and one basal fraction. Utilizing complete MCDB 153 basal nutrient culture medium containing epidermal growth factor and insulin, we then evaluated the clonogenic potential of cells in these three fractions. Suprabasal cell fractions demonstrated a colony-forming efficiency as great as or greater than that of the basal cell fraction, and 10%-15% of the keratinocytes that grew in primary and secondary cultures expressed involucrin, a suprabasal keratinocyte differentiation marker. Of such involucrin-containing keratinocytes, 80% also possessed the potential to undergo DNA synthesis, as determined by autoradiography following a 48-hour incubation with [3H]thymidine. These observations support the conclusion that the human keratinocytes that grow in vitro in serum-free medium can be derived from suprabasal cells and, therefore, that a state of nonterminal keratinocyte differentiation exists.

Two subtypes of reversible cell cycle restriction points exist in cultured normal human keratinocyte progenitor cells.

Three methods can induce reversible arrest of the growth of cultured human keratinocytes in the G1 phase of the cell cycle. These include the incubation of cells in medium containing transforming growth factor (TGF)-beta or ethionine, or in isoleucine-deficient medium. The current studies were performed to determine if the growth arrest induced by these methods occurs at a common or at a distinct G1 state(s). We first evaluated the relative time interval required for arrested cells to initiate DNA synthesis after growth restimulation with mitogenic medium. The results show that ethionine arrested cells require 22 to 28 hours to initiate DNA synthesis and that a maximum rate of DNA synthesis occurs at 46 hours. Cells arrested by isoleucine deficiency required 10 to 12 hours to initiate DNA synthesis with peak DNA synthesis occurring at 24 hours. Finally, TGF-beta arrested cells require only 6 to 8 hours to initiate DNA synthesis and show a maximum rate of DNA synthesis at 18 hours. We next evaluated if cells that were growth arrested at these states were differentially capable of initiating DNA synthesis in different types of potentially mitogenic medium. The results show that if TGF-beta arrested cells were refed TGB-beta free serum containing medium with ethionine or similar medium with isoleucine deficiency, no DNA synthesis occurred. In contrast, if cells whose growth was arrested in ethionine-containing medium or in isoleucine-deficient medium were refed mitogenic medium containing TGF-beta, significant DNA synthesis was detected. These results suggest that least two different types of reversible growth arrest states exist in cultured human keratinocytes. One appears to be mediated by receptor-dependent processes, such as that induced by TGF-beta, and the other appears to be mediated by other types of metabolic events, such as those induced by ethionine treatment or by isoleucine deficiency.

Biologic mechanisms for the regulation of normal human keratinocyte proliferation and differentiation.

Normal human keratinocytes can be grown in serum-free medium, and the integrated control of their proliferation and differentiation can be modulated experimentally. The growth of cultured human keratinocytes can also be specifically arrested at either reversible or irreversible growth arrest states. Reversible growth arrest is induced by culture in medium containing TGF-beta or ethionine or in medium deficient of isoleucine. Irreversible growth arrest is induced by culture in razoxane-containing medium or by routine passage of keratinocytes until senescence results. The current studies were performed to determine from which growth arrest states keratinocyte differentiation occurs. Cells were therefore growth-arrested at each state, and they were then incubated in several different differentiation-promoting culture conditions. The results show that differentiation, as determined by morphologic, cytochemical, and immunofluorescent assays, can be induced from multiple reversible and irreversible growth arrest states by a series of complex biologic mechanisms. More specifically, at least three distinct stages appear to be involved in the process of keratinocyte differentiation. First, cells arrest their growth at a reversible predifferentiation state. Second, cells irreversibly lose their proliferative potential. Finally, cells express the terminally differentiated keratinocyte phenotype.