DNA ploidy in nonmelanoma skin cancer.

BACKGROUND: Patients with head and neck squamous cell carcinoma die of locoregional recurrence and those with basal cell carcinoma suffer cosmetic and functional changes from its treatment. Prognostic factors are based upon tumor characteristics and host factors. Flow cytometry can assist with prognostic characterization of nonmelanoma skin cancer. METHODS: Specimens from 40 sequential patients with head and neck nonmelanoma skin cancers were prospectively obtained at the time of surgery. The patients were followed for four years for local recurrence and metastasis to regional lymph nodes. Samples were prepared from frozen specimens using a modification of the Vindelov procedure. RESULTS: DNA aneuploidy or tetraploidy and histology of well differentiated squamous cell carcinoma were significantly associated with metastasis to regional lymph nodes. Direct extension of tumor below the adipose tissue was associated with an S-phase greater than 4.1 and a proliferative fraction greater than 5.5. No tumors recurred at the site of surgical resection. CONCLUSIONS: The measurement of DNA ploidy of well differentiated squamous cell carcinoma, and proliferative capacity or S-phase of both basal cell and squamous cell carcinomas assists in predicting the biologic proclivity for locoregional invasion or metastasis of nonmelanoma skin cancer. Identification of aggressive tumors at the time of surgery may offer the opportunity for prevention of lethal metastasis by using adjunctive therapy.

Lipid composition and acid hydrolase content of lamellar granules of fetal rat epidermis.

Lipids and acid hydrolases have been characterized in a subcellular fraction, enriched with lamellar granules (LG), derived from fetal rat epidermis. This fraction contains 23% glycosyl ceramides and ceramides, 15% free sterols, and 34% phospholipids. The lipid/protein ratio is 2.0. The sterols and sphingolipids were present in proportions similar to those previously reported in stratum corneum. These findings provide direct biochemical evidence for the widely accepted hypothesis that stratum corneum lipids are derived from exocytosis of lamellar granules into the intercellular space. The LG fraction was enriched in certain acid hydrolases including glucosidase, acid phosphatase, phospholipases A, and sphingomyelinase; other acid hydrolases, i.e., amino-glycosidases, glactosidase and aryl sulfatase (pH 5.5), and steroid sulfatase were not preferentially localized in this fraction. By modulation of phospholipids, glycolipids, and proteins in the membrane regions of stratum corneum, the acid hydrolases of LG may play a role relevant to the function and desquamation of stratum corneum.

Sphingolipids of the stratum corneum and lamellar granules of fetal rat epidermis.

Sphingolipid profiles have been determined for whole epidermis, a subcellular fraction enriched in lamellar granules, and a fraction enriched with stratum corneum derived from fetal rat skin. In each case, 4 groups of glucosylceramides and 6 groups of ceramides have been identified by thin-layer chromatographic comparison with structurally defined sphingolipids from pig epidermis. The relative amounts of the sphingolipids in each preparation have been quantified by photodensitometry of the charred chromatograms. Lamellar granule sphingolipids had elevated proportions, relative to whole epidermis, of acylceramides, acylglucosylceramides, and a glucosylceramide fraction which may be produced by O-deacylation of the acylglucosylceramides. The fetal stratum corneum-enriched samples contain reduced proportions of all glucosylceramides and acylceramides as compared to lamellar granule lipids. The possible functions of these sphingolipids in the assembly and structure of lamellar granules are discussed.

Acid hydrolases of the epidermis: subcellular localization and relationship to cornification.

Three lysosomal-type acid hydrolases were examined in subcellular fractions of the developing epidermis of fetal rats to assess the relationship of degradative enzymes to cornification. As the granular layer developed and cornified between 18 and 20 days (D) of gestation, epidermal acid phosphatase increased, acid phospholipase A remained constant, and beta-glucuronidase activity declined. The enzymes were present in 3,000, 17,000, and 100,000 g particulate fractions and soluble cytoplasm. However distribution differed: acid phosphatase and phospholipase A were more preferentially localized than was glucuronidase in the 17,000 g fraction which excluded mitochondria and ribosomes and was enriched in lamellar granules. The findings suggested that acid phosphatase and phospholipase were present in membrane-bound organelles (e.g., lamellar granules) in the granular layer. Particulate acid phosphatase increased with granular layers on days 19 and 20 while a 7-fold increase in soluble enzyme coincided with cornification on day 20. As shown by isoelectric focusing, the enzyme became more heterogeneous at day 20 than at day 18, suggesting increased glycosylation. The particulate fraction displayed lysosomal characteristics with respect to release of acid phosphatase, which was inhibited by hydrocortisone and enhanced by retinol. When fetal epidermis was allowed to cornify in organ cultures, similar increases in acid phosphatase occurred. The presence of hydrocortisone did not affect increase in total enzyme but a greater proportion remained in the particulate fraction. The findings suggest that particulate acid phosphatase and phospholipase are compartmentalized in organelles with lysosomal characteristics during development of granular cells and that release of phosphatase is coincident with cornification. This may reflect not only exocytosis of lamellar granules but also intracellular release of the hydrolytic enzyme.

A method for partial purification of lamellar granules from fetal rat epidermis.

A subcellular fraction enriched with lamellar granules was obtained from homogenates of fetal rat epidermis by means of density gradient fractionation in metrizamide. 62% of organelles in this fraction were bounded by single membranes and measured 80-130 nm in the shorter diameter. About 10% of these had characteristic lamellae. Other structures in this fraction were larger vesicles (20%) or smaller organelles and vesicles (18%). The fraction had a low buoyant density (1.08-1.10) suggesting a high lipid content and contained a sharply localized peak of acid phosphatase activity. The 80-130 nm organelles reacted with bismuth after oxidation with periodic acid and were positive for acid phosphatase. Other intracellular organelles (e.g., lysosomes, mitochondria, ribosomes) as well as keratin fibrils and keratohyalin were not present. It is concluded that most of this fraction consists of lamellar granules, permitting for the first time detailed investigations of the composition and metabolism of these organelles.

The phospholipases A of epidermis.

Phospholipases A have been characterized in fetal rat epidermis. Both a calcium dependent phospholipase A with pH optimum of 8.5 and a calcium independent enzyme with a pH optimum of 4.5 were found. Activity against both acyl groups of phosphatidyl choline were found for both enzymes and the findings suggested that the acid enzyme (pH 4.5) is primarily phospholipase A1. The specificity of the alkaline enzyme with respect to acyl groups could not be determined due to the marked hydrolysis of lysophosphatidyl choline. Lysophospholipase activity had similar pH and calcium requirements as the alkaline phospholipase A and the 2 activities could not be dissociated in these experiments. The acid phospholipase was localized primarily in a fragile large particle fraction while the alkaline enzyme was present in various subcellular fractions and most active in the soluble supernatant. These findings demonstrate that epidermal phospholipases A are similar to those in other mammalian tissues. The presence of both acid and alkaline enzymes ar various subcellular loci suggest that the enzymes may play important roles in many facets of epidermal membrane metabolism and in particular in the degradative events of keratinization.

Flux of fatty acids during epidermal differentiation.

Fetal rat skin prior to cornification (18 days of gestation) was prelabeled with [14C] linoleic acid in vitro and subsequently allowed to differentiate and cornify in organ culture. During 48 hr in culture total epidermal 14C fatty acids and 14C dienes decreased in phospholipids and increased in triglycerides and sterol esters as granular layers and stratum corneum formed. During the second 24-hr period, there was no net loss in 14C fatty acids from the epidermis, suggesting that the changes in phospholipid and neutral esters represented translocations of fatty acids within the cornifying cells. The findings are consistent with the hypothesis that fatty acids liberated by hydrolysis of phospholipids are salvaged by reesterfication as neutral lipid esters during epidermal cornification.

Patterns of glucose catabolism during differentiation of fetal rat epidermis.

Glucose metabolism has been investigated during differentiation of fetal rat skin to evaluate shifting patterns in the catabolism of this major exogenous substrate in relation to changing requirements during epidermal maturation. Glucose utilization and aerobic glycolysis declined while oxidative metabolism increased in whole skin as stratification and cornification progressed. Activity of the hexose monophosphate shunt was quantitated in isolated epidermis for the first time. The relative activity of this pathway diminished somewhat from 20% to 12% of utilized glucose as the granular layer and stratum corneum developed. The results are consistent with (a) enhanced utilization of glucose, aerobic glycolysis, and hexose monophosphate activity in rapidly proliferating eidermis, and (b) flexibility of glucose catabolism in response to cellular requirements at different stages of differentiation.