Thymidine Labeling Index Research Articles

DNA flow cytometry and prognostic factors in 1331 frozen breast cancer specimens.

Breast cancer proliferative capacity as determined by the DNA thymidine labeling index, along with estrogen and progesterone receptor status, is highly predictive for risk of relapse and overall survival. Recently, DNA ploidy and proliferative capacity (S-phase fraction [SPF]) as determined by flow cytometry have also shown significant prognostic value. The authors have developed a technique which allows a 50 to 100 mg aliquot of the same frozen breast tumor specimen routinely employed in steroid receptor assays, to be assayed for both DNA ploidy and SPF by flow cytometry. Of the 1331 tumors examined, DNA histograms were evaluable for ploidy in 89% (1184) of specimens examined; 57% of these were aneuploid. Adapting a trapezoidal model to estimate SPF in both diploid and aneuploid tumors, the authors found 81% (1084) to be evaluable for SPF, with a median SPF of 5.8% for the entire population. The median SPF was significantly lower in diploid tumors (2.6%) than in aneuploid tumors (10.3%, P less than 0.0001). Both aneuploidy and high SPF were strongly associated with absence of steroid receptors. Aneuploid tumors showed more striking differences in the frequency of high S-phase values with respect to receptor status and age or menopausal status, whereas diploid but not aneuploid tumors showed lower SPF in node-negative versus node-positive patients. Because it is particularly important to identify the high-risk minority of node-negative patients, the authors examined the node-negative group separately. High SPF subgroups appeared in each category of receptor status and age or menopausal status within the node-negative group, suggesting that SPF will be an independent prognostic factor. With the DNA flow cytometric methods used here, it is now practical to determine ploidy and SPF for nearly every breast cancer patient. These factors, which show associations with established prognostic factors, such as receptor status can now be fully evaluated for their prognostic significance in broad patient populations.

Comparative analysis of [3H]-thymidine labelling index and monoclonal antibody Ki-67 in non-Hodgkin's lymphomas.

The relation between the [3H]-thymidine labelling index (3H-Thy LI), which evaluates the S-phase cells, and the monoclonal antibody Ki-67 (MoAb Ki-67), which recognizes a nuclear antigen expressed during the cell cycle, was defined in 35 non-Hodgkin's lymphomas (NHL). Significantly higher median values of [3H]-Thy LI and Ki-67-positive cells were observed for high-grade than for low-grade malignancy tumours according to the Kiel classification, but with wide and overlapping values for the two morphologic subgroups. A significant correlation was observed between [3H]-Thy LI and Ki-67-positive cells (P less than 0.0001; r = 0.62). However, the ratio between the two cell kinetic variables on individual tumours was not constant. It sharply increased with decreasing [3H]-Thy LI values and was much higher in low-grade NHL than in high-grade NHL. Follow-up studies are needed to establish the role of the tumour growth fraction as evaluated by MoAb Ki-67 as a prognostic indicator in NHL.

Influences of dihydrotestosterone, testosterone, estradiol, progesterone, or prolactin on the cell kinetics of human hyperplastic prostatic tissue in organ culture

In an effort to characterize the hormone sensitivity of human benign prostatic hyperplasia (BPH) maintained in organ cultures for 12-72 h, the influence of 5-alpha-dihydrotestosterone (DHT), testosterone (T), 17-beta-estradiol (E2), progesterone (Pg), or prolactin (PRL) was assessed on the cell proliferation rate of 25 BPH specimens by the use of tritiated thymidine incorporation followed by autoradiography. Significant increases in the thymidine-labeling index (TLI: percentage of labeled nuclei) were observed in glandular tissue after a 36-h incubation period in presence of DHT, E2, Pg, or PRL in 52%, 44%, 28%, and 60% of BPH cases, respectively. Nonparametric statistics (Spearman and Kendall rank correlation tests) have shown that 1) the steroid-induced TLI increases are dependent on the basal rate of cell proliferation, while the PRL-induced effect is independent of it, and 2) all the steroid-mediated effects on BPH TLI are correlated together, whereas they seem to be independent of the PRL-induced TLI increase. When T was compared with DHT on nine BPH specimens, three were found to be sensitive to both hormones, and two responded to DHT only. We propose that our study methods are suitable as a means to assess the hormone sensitivity of individual cases of BPH and possibly prostatic tumors.

Proliferative and secretory activity in the pregnant and lactating human breast.

In this study the proliferative and secretory activity of human pregnant and lactating breast has been examined. 17 cases of pregnant tissue were obtained, comprising 9 from first trimester, 6 from the second and 2 from the third, together with 3 cases of lactating breast. Proliferation, measured by 3H thymidine autoradiography, gave a constant Thymidine labelling index of approximately 6% during the first 20 weeks, with a reduction by 50% thereafter. Levels of 0.2% were seen during lactation. Morphological assessment showed a high frequency of both mitosis and apoptosis throughout gestation. Although mitotic frequency did reduce with time, values were more variable than thymidine labelling. The reduction in thymidine uptake halfway through pregnancy coincided with generalised morphological lactational differentiation and the major immunocytochemical expression of alpha-lactalbumin. However, cytoplasmic expression of the secretory component of IgA was generalised and marked even during the first trimester and was closely related to increased proliferation and general synthetic cellular activity. These and other observations support a complex relationship of autocrine and paracrine factors in addition to the customary endocrine ones for breast regulation.

Proliferative activity of rectal mucosa and soluble fecal bile acids in patients with normal colons and in patients with colonic polyps or cancer

Rectal biopsies and fecal collections were obtained from a consecutive series of 34 outpatients prior to colonoscopy at a gastroenterology clinic. Subsequently, 14 were found to have no colonic pathology, 13 had adenomatous polyps, (3 of those had a previous history of colon cancer) and 7 were diagnosed with colon cancer. In confirmation of earlier studies the tritiated thymidine labelling index was higher in patients with tumors than in those without pathology (7.9% vs. 5.8% with P = 0.06). The patients with colonic tumors also had significantly higher levels of deoxycholic acid ( P = 0.01) and lithocholic acid ( P = 0.005) in the aqueous extract of their feces. This study shows that these biochemical measures may indicate colon cancer risk.

Effects of sodium cholate on experimental carcinogenesis and cell proliferation in an excluded colonic segment.

Bile salts appear to be important promoters of colon carcinogenesis. This study was designed to assess the importance of the fecal stream in cholic acid-induced colon tumor promotion. Male Sprague-Dawley rats underwent transverse colostomy after induction with dimethylhydrazine (DMH) and the excluded distal colon was irrigated with saline or sodium cholate (23 microM) 5 times per week until sacrifice. Controls initially injected with saline were similarly treated. All surviving animals were sacrificed at 28 weeks after the last DMH injection. Five animals from each group were randomly chosen to assess tritiated thymidine labeling and distribution by autoradiography in normal appearing colon mucosa of irrigated bowel. Cholate irrigation failed to increase tumor yield or modify the proportion of adenomas and adenocarcinomas in this model. Paradoxically, fewer tumors per affected rat were noted with sodium cholate irrigation. Cholate irrigation also failed to affect crypt cellularity, thymidine labeling indices, and labeling distribution in DMH-treated rats and controls. An effect of DMH was seen, however, with an increase in thymidine labeling index and increased labeling in the top half of the crypt in all DMH-treated groups. This study suggests that tumor promotion with primary bile salts is not a direct affect and may result from further bile salt metabolism within the fecal stream. DMH-induced changes in cell proliferation were reproduced with this model. Use of an excluded colon segment to assess the effect of suspected tumor promoters on carcinogenesis or colon mucosal cell proliferation is feasible and may be a useful model for future studies.

Mechanisms of healing in synthetic grafts

In previous baboon studies we have shown that porous (60 μm mean internodal distance) polytetrafluoroethylene (PTFE) grafts heal by ingrowth of endothelium and smooth muscle cells from the adjacent artery and from capillaries penetrating through the interstices of the graft. However, porous grafts (principally made of Dacron) in humans do not heal. This has been attributed to a wound healing deficiency in humans; however, it might be due to an inhibitory effect of the Dacron itself. To examine the latter possibility, we undertook this study to compare the healing of 4 mm internal diameter porous Dacron grafts (USCI, Sauvage Filamentous Knitted) with that of Gore-Tex 60 μm PTFE grafts in baboons (the latter graft not available for clinical use). The grafts were harvested at 2, 4, and 12 weeks and assessed for (1) percentage of endothelial coverage, (2) endothelial cell (EC) proliferation (thymidine labeling index), (3) intimal area, and (4) smooth muscle cell (SMC) proliferation (thymidine labeling index). The PTFE grafts at all three time points were fully covered, whereas only one of five Dacron grafts was completely covered at 12 weeks. The intima of the PTFE grafts consisted of ECs and SMCs, whereas that of the Dacron grafts contained ECs and SMCs as well as focal accumulations of thrombus. The intimal cross-sectional areas in the Dacron grafts (3.0 ± 1.2 mm2) were significantly greater than in the PTFE grafts (0.8 ± 0.6 mm2) at 4 weeks; there was no difference at 12 weeks (Dacron, 2.6 ± 2.3 mm2 and PTFE, 3.0 ± 2.5 mm2). SMCs in the intima of the Dacron and PTFE grafts were actively proliferating at 4 weeks but returned to normal levels at 12 weeks. ECs continued to proliferate even at 12 weeks in both Dacron and PTFE grafts, suggesting continued denudation and replacement. Although both USCI Dacron and porous PTFE grafts heal by proliferation of cells derived from invading capillaries, nevertheless endothelial coverage in some Dacron grafts is incomplete even as late as 12 weeks. This result suggests that the failure of Dacron grafts in humans to develop an endothelial lining might be attributable in part to the graft material itself.

Immunofluorescence Labeling Indices in Myeloma and Related Monoclonal Gammopathies

We measured plasma cell labeling indices (LI) in 52 patients with monoclonal gammopathies. Cytoplasmic reactivity with polyspecific or kappa- and lambda-specific light chain anti-Ig reagents identified monoclonal plasma cells, plasmablasts, and lymphocytoid plasma cells. Among newly diagnosed untreated patients, a high immunofluorescence LI distinguished those with multiple myeloma (MM) from those with stable monoclonal gammopathies (P less than 0.002). Among treated patients with MM, those in the plateau phase of the disease had low LI, whereas patients in the relapse phase or early in treatment had high LI. The immunofluorescence LI correctly classified three more patients with newly diagnosed MM than did the tritiated thymidine LI technique. LI specific for the neoplastic plasma cells resulted in excellent discrimination of patients with active disease. Because results are easily and rapidly obtained, this technique is useful clinically.

Effect of ovariectomy, hypophysectomy and/or GnRH analog (HRF) administration on the cell proliferation of the MXT mouse hormone-dependent mammary tumor

The MXT tumor is an experimental mammary neoplasm which is maintained by serial transplantation using B6D2F1 mice, and which contains significant amounts of estrogen and progesterone receptors. The aim of the present study is to examine the effects of ovariectomy (OVX) or ovariectomy plus hypophysectomy (OVX-HX) on both the macroscopic growth and the cell proliferation of this tumor. This cell proliferation was evaluated by means of in vivo tritiated thymidine autoradiography. In addition, we investigated the effects of a GnRH analog (Gonadorelin: HRF, 5-oxo-Pro-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-hydrochloride) on MTX tumor cell proliferation on 7 day-OVX and 5 day-HX (OVX-HX) mice. The uterine luminal epithelium was chosen to monitor the methodology. Our data clearly demonstrate that there is a delay in the growth of MXT tumors grafted into hypophysectomized animals and, to a lesser degree, ovariectomized animals. With respect to proliferation, castration induced a dramatic decrease of the thymidine labelling index (TLI) in the tissue used to monitor the methodology (the uterine luminal epithemium); in contrast, no cell proliferation was induced by hypophysetomy or HRF administration. In 4-week-old MXT tumors, ovariectomy also markedly decreased the TLI within a few days of its taking place. However hypophysectomy, performed on castrated animals, induced a significant and transient increase of cell proliferation in this neoplasm, an increase which lasted from the 2nd to the 5th day following the operation. The high basal level of MXT cell proliferation recorded in OVX-HX animals decreased dramatically after the administration of HRF between 12 and 48 h prior to the sacrifice of the animals. It is concluded that the HRF exerts a direct effect on the MXT tumor cells, and this HRF might be essential for their growth.

Hepatic adenoma within a spindle cell carcinoma in a woman with a long history of oral contraceptives

The case of a young woman is presented who had a long history of oral contraceptive use and who was found to have a focal hepatic adenoma within a large spindle cell carcinoma of the liver. The pleomorphic tumor is positive for keratin and alpha 1-antitrypsin by immunohistochemistry, has intracellular lumina by electron microscopy, a moderately high thymidine labeling index, and is negative for estrogen and progesterone receptors. A review of the literature concerning liver tumors occurring in young patients indicates that there is an underlying disturbance of the hormonal milieu in all instances reported. The prognosis is widely variable and unpredictable.

Influence of systematic errors on the evaluation of the S phase portions from DNA distributions of solid tumors as shown for 328 breast carcinomas.

The portion of cells in S phase has proved to be a valuable prognostic indicator of early relapse and life expectancy, particularly in breast carcinoma. Comparisons of published data on samples of primary breast carcinoma biopsies showed that the values obtained by analyses of flow cytometric DNA distributions were generally higher than those of determinations based on the tritiated thymidine (3H-ThdR) labeling index (LI). Flow cytometric DNA analyses of 328 biopsy samples of primary breast carcinomas revealed that these differences could be explained by varying contributions of debris background. Since this influence is inversely proportional to the cell counts in each channel, it may cause considerable errors, particularly in the S phase channels, which normally contain the lowest counts of the DNA distributions. Two different mathematical rationales were tested in order to separate DNA distributions from the debris superimposition. No appreciable differences were found with respect to the essential results. After appropriate subtraction of the background levels, the previously reported discrepancies between cytometrically determined S phase portions and 3H-ThdR LI values disappeared, and good agreement was achieved for the comparable tumor samples of the present study. In conclusion, debris background subtractions should generally precede the DNA histogram analyses, particularly of solid tumors, in order to obtain reliable S phase values.

Assessment of tumor cell kinetics by immunohistochemistry in carcinoma of breast.

Cell proliferation was assessed in 33 invasive breast carcinomas by an immunoperoxidase procedure using the monoclonal antibody, Ki-67, which reacts with a nuclear antigen in proliferating cells. The antibody labeled a variable proportion of tumor cells ranging from 3% to 60%. High numbers of Ki-67-positive cells were found in tumors with high mitotic rates, high nuclear grade, high histologic grade, and in premenopausal women. Tumors with low and intermediate Ki-67 labeling rates often had high estrogen receptor content, whereas tumors with high Ki-67 labeling rates were usually estrogen receptor negative. These correlations are similar to those previously reported for other measurements of cell cycle kinetics such as thymidine labeling index and suggest that immunohistochemical staining of invasive breast carcinoma for the Ki-67 epitope may provide cell cycle information not otherwise readily available to the clinician and may be useful in assessing prognosis in carcinoma of the breast.

Survival following combination chemotherapy in advanced high grade non-Hodgkin's lymphomas: relation to proliferative activity of the lymphoma cells.

In 18 untreated adult patients (median age 62.5 yr) with advanced non-Hodgkin's lymphoma of unfavourable histology, thymidine labelling indices (LIs) of the lymphoma cells were assessed. The patients were treated with combination chemotherapy and have been followed for 29-60 (median 52) months or until death. The survival curve had a steep fall during the first 2 yr. Between 2-5 yr after treatment there was a flattening of the curve and survival seemed to be similar to the survival expected for a Swedish population matched for age and sex. 11 patients died with 2 yr and 7 patients have survived for a longer period. Age, histopathologic classification and clinical stages were comparable in short-term and long-term survivors and treatment was not more aggressive for the long-term survivors. The LIs were significantly higher (median 8.2) in short-term survivors than in the long-term survivors (median 1.4). Long-term survival following combination chemotherapy of advanced NHL of unfavourable histology seems to be achieved mainly in patients with a low proliferative activity of the lymphoma cells. It is suggested that in NHL a high proliferative activity may facilitate the generation of new mutants and that some of these are spontaneously resistant to various chemotherapeutic drugs.

Cell proliferation kinetics of cultured human keratinocytes and fibroblasts measured using a monoclonal antibody

Measurements of cell cycle kinetics using an immunoperoxidase method employing monoclonal anti-bromodeoxyuridine (BrdU) antibody were compared with an autoradiographic method using [3H]-thymidine. The methods were applied to epithelial cells grown from explants of normal skin and basal cell carcinoma (BCC), and to fibroblasts from normal skin, hypertrophic scar and keloid. In normal keratinocytes the number of peroxidase-positive cells was higher than the number incorporating [3H]-thymidine, because of the presence of labelled cells in the centre of the explants in the former. The epithelial cells from BCC gave a mean (+/- SD) of 4.9 +/- 1.2% peroxidase-positive cells, while no cells were labelled with [3H]-thymidine. In dermal fibroblasts from normal skin and hypertrophic scar the percentages of peroxidase-positive cells did not differ significantly from the [3H]-thymidine labelling indices. The immunological method has advantages over [3H]-thymidine autoradiography in that it avoids radioactive material and the proportion of cells labelled by the two methods is the same.

Mitotic activity in non-Hodgkin's lymphoma. Relation to the Kiel classification and to prognosis.

At histopathological diagnosis of non-Hodgkin's lymphoma (NHL) the mean number of mitoses in 10 high power fields (X 40) was determined in thin sections (2 micron) and designated 'mitotic index' (MI). In 38 patients the thymidine labelling index (LI) of the lymphoma cells was also determined. There was a close correlation between MIs and LIs (r = 0.81, P less than 0.001) indicating that MI reflects the proliferative activity in NHL. Among 101 patients with NHL classified according to the Kiel nomenclature MIs were generally lower in lymphomas of low grade malignant type than in the high grade malignant lymphomas. The variation of MIs within morphological subgroups was especially pronounced in high grade lymphomas. Only 18 of 49 patients (37%) with MI greater than or equal to 2 have survived for 2 years in contrast to 37 of 52 patients (77%) with MI less than 2 (P = 0.001). For patients with histologically low grade lymphomas and MI greater than or equal to 2.0 the median survival was 23 months and for those with MI less than 2.0 58 months (P = 0.09). Patients with high grade lymphomas and MI greater than or equal to 2.0 had a median survival of 15 months compared to 57 months for those with MI less than 2.0 (P = 0.04). In a multivariate analysis of 50 patients with centroblastic-centrocytic (CB-CC) or centroblastic (CB) lymphomas the importance of different prognostic factors was analysed. Among the variables age, MI, growth pattern (follicular vs. diffuse), cell type (CB-CC vs. CB), clinical stage (I vs. II-IV), initial chemotherapy (active vs. less active) only age and MI gave significant prognostic information. It is concluded that the assessment of mitoses in NHL gives prognostic information in addition to histopathologic classification. The method is simple and the proliferative activity and histopathological diagnosis can be ascertained routinely on the same occasion.

Subchronic (13‐week) inhalation toxicity study of formaldehyde in rats

Male and female albino Wistar rats were exposed to concentrations of 0, 1, 10 or 20 ppm formaldehyde vapour during 6 h/day, 5 days/wk for 13 weeks. Treatment-related changes observed at 20 ppm included in both sexes: stared coats, uncoordinated locomotion and excitation during the first 30 minutes of each exposure, yellowing of the fur, growth retardation, a decreased level of plasma protein, severe and extensive karatinized stratified squamous metaplasia of the nasal respiratory epithelium, and focal degeneration and squamous metaplasia occasionally accompanied by keratinization of the olfactory epithelium; in males only; increased activities of plasma aspartate amino transferase (ASAT), alanine amino transferase (ALAT) and alkaline phosphatase (ALP) and squamous metaplasia of the laryngeal epithelium. Lesions seen at 10 ppm included yellowing of the fur and moderate squamous metaplasia of the nasal respiratory epithelium. The only change observed in three out of twenty 1 ppm exposed animals that might or might not be treatment-related was minimal focal epithelial hyperplasia and squamous metaplasia of the respiratory epithelium lining the nasal septum and maxillary turbinates. No histopathological evidence of hepatotoxicity was detected in any of the formaldehyde-treated groups. An in vivo/in vitro cell proliferation study showed an increase in [3H]-thymidine labeling index of the respiratory epithelium lining the nasoturbinates of rats exposed to 10 or 20 ppm formaldehyde on three successive days, whereas at the 1 ppm level the labeling index was similar to that of controls. It was concluded that under the conditions of the present 13-week inhalation study, formaldehyde at concentrations up to 10 ppm was not hepatotoxic to rats. At the 20 ppm formaldehyde level, a slight effect on the liver of male rats cannot be completely excluded. The study was inconclusive with respect to 1 ppm formaldehyde being a cytotoxic or a no-cytotoxic effect level for the nasal epithelium.

Cardiac hypertrophy in chick embryos induced by hypothermia.

A decrease in incubation temperature from 38 to 32 degrees C elicits a decrease in chicken embyro size and weight with concomitant heart enlargement if done after day 10 of incubation. When assayed at day 18 of incubation with the hypothermia started on day 11 or 14, evidence is presented that the heart enlargement is an hypertrophy with no detectable hyperplasia. Supporting data are presented for various physical parameters showing increases in heart wet and dry weight, volume, area, wall thickness, and cell size. There was little difference in DNA content and nuclear [3H]thymidine labeling index between hearts of control and hypothermic embryos. Hearts of hypothermic embryos showed a slight increase in water content and considerable increases in RNA, protein, and glycogen content per unit DNA. The average size of polysomes isolated from hypothermic hearts was larger than that of polysomes isolated from controls. Microscopic studies showed no obvious increase in amount of capillary beds, connective tissue, and myocardial cells. Annulate lamellae were found only in myocardial cells of hypothermic embryos in sparse amounts and low frequency but always associated with large deposits of glycogen.

Kinetics of vein graft hyperplasia: Association with tangential stress

Vein grafts become thicker as they adapt to arterial circulation, and in large measure this is the consequence of cellular hyperplasia. The kinetics of smooth muscle cell (SMC) and endothelial cell (EC) replication were evaluated in rabbit jugular vein segments transplanted into the carotid arterial circulation to define the role of cell proliferation in this process, and these data were correlated with dimensional changes in grafts from 1 hour to 24 weeks after implantation. At 1 hour most of the perianastomotic endothelium was denuded, as were occasional cells in the graft away from the anastomosis. EC replication (thymidine labeling index) in the center of the graft increased 400 times during the first week and produced an intact endothelial surface by 2 weeks. The EC thymidine index then fell progressively to quiescent pretransplant levels by the twelfth week. SMC replication was maximal at 1 week after grafting and returned to near quiescent levels at 12 and 24 weeks. Graft wall thickness and cross-sectional area reached a maximum at 12 weeks and then remained constant. Deoxyribonucleic acid content exhibited a thirty-five-fold increase in the first 4 weeks, then did not change. Likewise, SMC mass did not increase after 4 weeks. Further increase in cross-sectional wall area between 4 and 12 weeks was accounted for by accumulation of connective tissue. The final ratio of luminal radius to wall thickness decreased to a level equal to that of normal artery. Since blood pressure did not change over the period of the study, this observation supports a possible regulatory role for wall tangential stress in the modification of vein graft structure after transplantation into the arterial circulation.

Estrogen receptor status, histologic tumor grade and thymidine labeling index in primary breast carcinoma

Estrogen receptor status, histologic tumor grade and thymidine labeling index in primary breast carcinoma

Response of Carcinogen-Altered Mouse Epidermal Cells to Phorbol Ester Tumor Promoters and Calcium

Primary cultures of mouse epidermal cells are induced to terminally differentiate when extracellular calcium levels are increased to more than 0.1 mM. After carcinogen treatment, cellular foci can be selected that resist this calcium signal to terminally differentiate. Calcium causes these foci to stratify; however, in contrast to normal epidermis, DNA-synthesizing cells in these foci are found in the suprabasal cell layers as well as in basal cells. Cell lines derived from these foci may be considered to be putative initiated cells. Three of these cell lines, designated 308, D, and F, have been characterized for their response to calcium and phorbol ester tumor promoters. The formation of cornified cells and the activity of epidermal transglutaminase were utilized as markers of epidermal differentiation. Neither calcium nor the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increased transglutaminase activity or cornification of any of the 3 lines. Proliferation was estimated by the [3H]thymidine labeling index, by incorporation of [3H]thymidine into DNA, and by a clonal growth assay. Unlike primary normal cultures, raising the calcium level of the medium did not markedly reduce the rate of proliferation of any of the 3 cell lines. In 2 of the lines, line 308 and line D, proliferation increased in response to TPA exposure. In line F, [3H]thymidine incorporation in confluent cultures was inhibited by TPA, while in cells plated at clonal densities, TPA was cytotoxic at doses of 5 ng/ml or higher. If these calcium-resistant epidermal cell lines correspond to initiated cells, their lack of sensitivity to the induction of terminal differentiation by TPA could account for their growth relative to normal cells. Those lines that also respond to stimulation of proliferation by TPA to a greater extent than normal cells would have a further growth advantage.