Placental infection with human papillomavirus is associated with spontaneous preterm delivery.

BACKGROUND: We sought to determine if human papillomavirus (HPV) infection of extravillous trophoblast cells reduces cell invasion and if placental infection is associated with adverse reproductive outcomes attributed to placental dysfunction. METHODS: We conducted apoptosis and invasion assays using extravillous trophoblast (HTR-8/SVneo) cells that were transfected with a plasmid (pAT-HPV-16) containing the entire HPV-16 genome. In order to associate HPV infection with reproductive outcomes, we conducted a case-control study to detect HPV DNA in the extravillous trophoblast region of placentas from cases of spontaneous preterm delivery, severe pre-eclampsia requiring delivery at <37 weeks and controls who delivered at term. RESULTS: Rates of apoptosis were 3- to 6-fold greater in transfected cells than in non-transfected cells or cells transfected with an empty plasmid. Invasion of transfected cells through extracellular matrices was 25-58% lower than that of the controls. HPV was detected more frequently in placentas from spontaneous preterm deliveries than in placentas from controls (P = 0.03). Identification of HPV in placentas from cases of pre-eclampsia was not significantly different to controls. CONCLUSIONS: HPV infection of extravillous trophoblast induces cell death and may reduce placental invasion into the uterine wall. Thus, HPV infection may cause placental dysfunction and is associated with adverse pregnancy outcomes, including spontaneous preterm delivery.

Adverse reproductive outcomes in urban women with adeno-associated virus-2 infections in early pregnancy.

BACKGROUND: We demonstrated recently that adeno-associated virus-2 (AAV-2) DNA was detected significantly more frequently in placental trophoblast cells from cases of severe pre-eclampsia than from normal term deliveries. Here, we sought to determine if maternal AAV-2 infection early in pregnancy preceded adverse outcomes resulting from placental dysfunction. METHODS: We collected first trimester maternal serum samples and compared anti-AAV-2 IgM antibody levels (indicating primary infection or reactivation of latent AAV-2) between controls delivered at term (n = 106) and three groups of cases: spontaneous abortions (n = 34), spontaneous preterm deliveries (n = 24) and women with at least one outcome usually attributed to placental dysfunction, including pre-eclampsia, intrauterine growth restriction (IUGR) or stillbirth (n = 20). The seroprevalence of immunoglobulin G (IgG) antibodies against AAV-2 and IgM antibodies against viruses that promote AAV-2 replication [adenovirus and cytomegalovirus (CMV)] were also determined. RESULTS: First trimester maternal IgM seropositivity was 5.6 times more prevalent among pre-eclampsia/IUGR/stillbirth cases (P = 0.0004) and 7.6 times more prevalent among preterm deliveries (P < 0.0001) than among controls. CMV and adenovirus IgM antibodies and chronic AAV-2 infections (IgG seropositivity) were not associated with adverse pregnancy outcomes. CONCLUSIONS: Primary or reactivated AAV-2 infection (maternal IgM seropositivity) early in pregnancy was associated with adverse reproductive outcomes associated with placental dysfunction, including pre-eclampsia, stillbirth and spontaneous preterm delivery.

Deep vein thrombosis: prevalence and risk factors in rehabilitation admissions with brain injury.

OBJECTIVE: To determine the prevalence and risk factors of deep vein thrombosis (DVT) among neurorehabilitation admissions with acquired brain injury (BI). METHODS: In this prospective, sequential case series, 709 consecutive initial neurorehabilitation patients with BI < 120 days-including traumatic brain injury (TBI; n = 360), intracranial hemorrhage (ICH; n = 213), primary brain tumor (n = 66), and hypoxia/other BI (n = 70)--were screened for evidence of DVT with lower extremity venous duplex ultrasonography (VDU). The admission screening protocol combined VDU and a commercial d-dimer (Dimertest [DDLx]) latex agglutination assay. DVT was considered present based upon VDU results only. RESULTS: DVT prevalence was 11.1%, and was higher with brain tumor (21.2%) and ICH (16%) than with TBI (6.7%) (chi2 test; p = 0.001). DVT risk factors identified by multivariable logistic regression analysis in the overall sample included older age (p = 0.002), type of BI (p = 0.04), DDLx (p = 0.0001), and greater postinjury duration (p = 0.0001), with a trend observed regarding lower Functional Independence Measure (FIM) locomotion (FIM-Loco) subscale score (p = 0.07). However, risk factors also varied with type of BI. Among patients with TBI, only DDLx (p = 0.001) and greater postinjury duration (p = 0.001) were associated with DVT. CONCLUSIONS: Admission venous duplex ultrasonography revealed occult proximal lower extremity deep vein thrombosis in 11% of neurorehabilitation patients with acquired brain injury. Deep vein thrombosis risk is multifactorial in this heterogenous patient population, with relative factor risk influenced by type of acquired brain injury. Semiquantitative d-dimer latex agglutination assay correlated significantly with presence of deep vein thrombosis.

Reoperation for neurological complications following carotid endarterectomy.

BACKGROUND: There remains a dilemma whether or not to re-explore the carotid artery when a neurological complication occurs after carotid endarterectomy. This study reviewed the indications for, findings and clinical outcomes following re-exploration. METHODS: Patients who experienced transient or permanent neurological events following carotid endarterectomy were identified from a prospectively compiled computerized database. Case notes were retrieved to determine time to onset of symptoms, use of carotid artery imaging and details about patients who had surgical re-exploration, and outcomes. RESULTS: Some 780 consecutive carotid endarterectomies were performed over 16 years, with an incidence of major stroke or death of 2.3 per cent (18 patients). Fifty-one patients experienced transient or permanent neurological events following surgery, 25 of whom underwent re-exploration. The findings included carotid thrombosis (ten patients), flap or other technical cause (three), haematoma (two) and no abnormality (ten). The neurological outcome after 30 days was similar, whether or not the carotid artery was re-explored. CONCLUSION: Carotid artery re-exploration was undertaken in approximately half of the patients who developed neurological complications following carotid endarterectomy. Although the cause was identified and a secondary procedure was undertaken in 14 of 25 patients, there was no improvement in clinical outcome at 30 days compared with that of patients managed non-operatively.

Evaluation of mild-to-moderate dysplasia on cervical-endocervical (Pap) smear: a subgroup of patients who bridge LSIL and HSIL.

The Bethesda System recommends Pap smear diagnosis to be based on the most abnormal cells present, regardless of number. Our reporting system includes a diagnostic category of mild-to-moderate dysplasia (D1-2), defined as cases with only a few moderately dysplastic cells. We evaluated the validity of a D1-2 diagnostic category by reevaluation of 58 cases with subsequent follow-up (up to 24 months). On biopsy and/or Pap smear follow-up, 24 cases (41%) showed LSIL and 34 cases (59%) showed HSIL. Index smears from these cases were examined by two cytopathologists blinded to patient follow-up for the following morphologic features: volumes (scale 1-4) of LSIL, HSIL, and dyskeratosis, HSIL as single cells or syncytial fragments, and acute inflammation. None of the morphologic features evaluated were significantly different between the LSIL and HSIL follow-up groups based on univariate and multivariate logistic regression analysis. The diagnosis of D1-2 on Pap smear is a valid diagnostic category that defines a subgroup of patients with both LSIL and HSIL follow-up, which cannot be reliably predicted based on morphology alone.

Follow-up of morphologically reactive lymphoid proliferations in fine-needle aspirates of elderly patients.

Fine-needle aspiration (FNA) is an effective tool in evaluating the cause of lymphadenopathy. While the morphologic diagnosis of a reactive lymphoid proliferation is common in younger patients, this diagnosis should be made carefully in older patients (those over the age of 50 yr) in light of the facts that such purely reactive conditions occur much less frequently in this population, and that follow-up of these patients reveals a malignancy (usually lymphoma) in a significant number of cases. In this series, we identified 40 patients with a morphologic diagnosis of reactive lymphoid proliferation on FNA and obtained their follow-up information. Of 19 patients under the age of 50 yr, 5 underwent subsequent biopsies and only one revealed a definitive malignancy (5%). In contrast, 7 of 21 patients over the age of 50 yr underwent a subsequent biopsy, and 6 were found to have a malignancy (5 malignant lymphomas, 1 metastatic melanoma). The higher rate of positive follow-up (29%) in this age group supports previous suggestions that morphologically reactive (mixed) lymphoid proliferations be viewed with increased suspicion in the elderly patient, and that additional studies, such as flow cytometry, be performed when material is available.

Lobectomy with tangential pulmonary artery resection without regard to pulmonary function.

BACKGROUND: Non-small cell carcinoma of the lung invading the pulmonary artery (PA) has traditionally been treated by pneumonectomy. Although PA resection and reconstruction (PAR) has begun to gain acceptance, previous series of PAR by the simplest technique of tangential excision and primary repair have been unfavorable. We have maintained a policy of performing PAR preferentially whenever anatomically feasible, and usually this has been possible by tangential excision and primary repair. This study sought to determine if this approach is sound. METHODS: Retrospective clinical and pathologic review. RESULTS: Thirty-three PARs were performed from 1992 to 1999. The patients, followed 6 to 65 months (mean 25), were aged 36 to 80 years (mean 61), and their tumors were pathologic stage IB (n = 7), IIB (n = 13), IIIA (n = 9), and IIIB (n = 4). The mean preoperative forced expiratory volume in 1 second was 70% predicted. The procedures included 14 bronchial sleeve lobectomies with PAR and 19 simple lobectomies with PAR. The PARs were performed without heparinization and included 19 tangential excisions with primary closure, 11 larger tangential excisions with pericardial patch closure, and 3 sleeve resections. There were no operative deaths and 2 (6.1%) early major complications, all unrelated to the PAR. Thirteen patients (39%) had early minor complications. Four-year Kaplan-Meier survival was 48.3% for stages I/II and 45% for stage III. Ipsilateral, central, intrathoracic recurrence occurred in 3 patients (9.1%). CONCLUSIONS: These data are not dramatically different from those reported for standard resections. Although the numbers are small, the results suggest that lobectomy with PAR by tangential excision is an acceptable alternative to pneumonectomy whenever anatomically possible.

Duodenal carcinoid tumor: report of a case diagnosed by endoscopic ultrasound-guided fine-needle aspiration biopsy with immunocytochemical correlation.

Fine-needle aspiration biopsy is a reliable and accurate method for the endoscopic diagnosis of gastrointestinal malignancies and it is particularly well suited for evaluation of submucosal lesions. We report the cytopathologic findings of a case of malignant carcinoid tumor of a 44-year-old male who presented with melena and a nonhealing duodenal ulcer. Endoscopic ultrasound examination revealed a submucosal lesion in the pyloric region. Fine-needle aspiration revealed abundant cellularity with tumor cells arranged in sheets and loose groups and dispersed single cells in a clean background. Papillary fragments, capillaries cuffed by tumor cells, and rosette formation were also noted. The cells were moderate in size, round to oval, with a small subpopulation of spindle-shaped cells. The nuclei were uniform, round to oval, with smooth nuclear borders. The chromatin pattern was finely granular with a salt-and-pepper appearance. The cytoplasm of the cells was small to moderate in amount, pale, and showed fine granularity. The differential diagnosis included a neuroendocrine neoplasm vs. an epithelioid gastrointestinal stromal tumor. The tumor cells were focally positive for chromogranin and negative for CD34, supporting the diagnosis of a neuroendocrine neoplasm. The differential diagnosis of primary gastrointestinal carcinoid tumors from gastrointestinal stromal tumors can be very difficult in cytologic material. In cases when diagnostic material is scant, or only present on one smear, the use of smear division and cell transfer in order to perform immunocytochemical stains may be of considerable value to confirm the neuroendocrine nature of the neoplasms.

Ethylene glycol poisoning: toxicokinetic and analytical factors affecting laboratory diagnosis.

Ethylene glycol poisoning is an important toxicological problem in medical practice because early diagnosis and treatment can prevent considerable morbidity and mortality. When ingested in the form of antifreeze or other automotive products, ethylene glycol results in central nervous system depression, cardiopulmonary compromise, and renal insufficiency. Metabolism of ethylene glycol to organic acids is required for metabolic derangement and organ damage. Laboratory features of ethylene glycol poisoning include increased anion gap metabolic acidosis, increased osmolal gap, calcium oxalate crystalluria, and detectable ethylene glycol in serum. This Case Conference integrates discussion of the toxicokinetic and analytical variables that affect the laboratory diagnosis of ethylene glycol intoxication.

Hematologic abnormalities occur in both cortical and lacunar infarction.

BACKGROUND AND PURPOSE: Primary hematologic abnormalities are a rare but established cause of ischemic stroke. In addition, activation of hemostatic parameters is often present during the acute phase of stroke. However, it is uncertain whether these abnormalities occur in both cortical and lacunar infarction; this study aimed to further assess this issue. METHODS: Hematologic parameters (prothrombin, activated partial thromboplastin, thrombin clotting, and euglobulin lysis times; and fibrinogen, fibrinopeptide A, antithrombin III, protein C, protein S, and plasminogen levels) were measured in 19 patients within 48 hours of the onset of acute cerebral infarction. These patients included 10 with cortical infarcts and 9 with lacunar infarcts, as determined by standard clinical and radiological criteria. RESULTS: Five patients with lacunar infarction and 7 patients with cortical infarction demonstrated raised fibrinopeptide A levels, indicating enhanced thrombin activity. Fibrinolysis, assessed by the euglobulin lysis time, was impaired in 6 of 9 patients with lacunar infarction and in 2 of 10 patients with cortical infarction. Lupus anticoagulants were detected in 3 patients with lacunar infarction and in 1 patient with cortical infarction. Three patients in each group displayed decreased antithrombin III function, and 1 patient with a lacunar infarction had a low protein C level. CONCLUSIONS: Primary hematologic disorders and secondary hemostatic derangements may occur in patients with either cortical or lacunar infarction.

Cytometrically coherent transfer of receptor proteins on microporous membranes.

A new method (Freeze-Transfer) is described for performing high-resolution immunocytochemistry for soluble cell proteins on frozen sections of biological tissues that involves thaw-mounting frozen tissue sections directly onto the surface of nitrocellulose thin films instead of directly onto glass slides. This technically straight-forward change in methodology resulted in chromogenic immunocytochemical assays for Her-2 and EGF receptors that were 1-2 orders of magnitude more sensitive while still fully utilizing the diagnostic resolving power of light microscopy. The effects of membrane pore size and surface chemistry on the resolution and intensity of Her-2 signal suggest that the enhanced sensitivity of Freeze-Transfer was caused by the cytologically coherent transfer of target molecules normally lost from cut surfaces of cells mounted on nonporous glass during assay.

Characterization of Int-5, a locus associated with early events in mammary carcinogenesis.

Three chemically-induced precancerous mammary hyperplasias, independently isolated in BALB/c mice, all contained mouse mammary tumor virus (MMTV) proviral DNA integrated into a common region in chromosomal DNA, designated Int-5 (Formerly Int-H, Gray et al., 1986). This site was cloned from a hyperplastic outgrowth (D2) into lambda phage. A 1.7 Kb Hind III DNA fragment, which flanks the 5' end of the MMTV insert, was generated from the cloned Int-5 region. This fragment was used as probe (IH-2) to localize Int-5 to mouse chromosome 9. The IH-2 sequence was highly conserved in DNA of several mammalian species including man, in three other widely divergent vertebrate phyla, and in C. elegans. The Int-5 region, containing 5.6 Kb 5' and 12.8 Kb 3' to the MMTV integration site in D2, was cloned in EMBL-3 from a BALB/c genomic DNA library. cDNA complementary to poly A+ lactating mammary gland RNA, annealed with Sst-1 fragments spanning most of the BALB/c Int-5 clone. The highest level of Int-5 specific poly A+ mRNA was detected in D2 tumor. Lactating mammary gland and D2 hyperplastic alveolar nodule contained 5-fold less Int-5 RNA while liver contained 8-fold less Int-5 RNA. Int-5 cDNA (IH-3) annealed with two RNA species of approximately 3.3 and 4.0 Kb. These data are consistent with the hypothesis that Int-5 contains an oncogene, different from any other previously described, involved in early events in some models of chemical carcinogenesis.

Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10.

Two sublines of a breast epithelial cell culture, MCF-10, derived from human fibrocystic mammary tissue exhibit immortality after extended cultivation in low calcium concentrations (0.03-0.06 mM) and floating transfers in low calcium (MCF-10F), or by trypsin-Versene passages in the customary (normal) calcium levels, 1.05 mM (MCF-10A). Both sublines have been maintained as separate entities after 2.3 years (849 days) in vitro and at present have been in culture for longer than 4 years. MCF-10 has the characteristics of normal breast epithelium by the following criteria: (a) lack of tumorigenicity in nude mice; (b) three-dimensional growth in collagen; (c) growth in culture that is controlled by hormones and growth factors; (d) lack of anchorage-independent growth; and (e) dome formation in confluent cultures. Cytogenetic analysis prior to immortalization showed normal diploid cells; although later passages showed minimal rearrangement and near-diploidy, the immortal cells were not karyotypically normal. The emergence of an immortal culture in normal calcium media was not an inherent characteristic of the original tissue from which MCF-10 was derived since reactivated cryo-preserved cells from cultures grown for 0.3 and 1.2 years in low calcium were incapable of sustained growth in normal calcium.

The use of restriction fragment polymorphisms to identify the cell line MCF-7.

We have recently documented variations in the level of N-ras gene amplification in independently passaged MCF-7 cells. To establish if these differences are due to a lack of genetic identity between current MCF-7 passages and the original cell line, we have performed karyological and restriction fragment length polymorphism (RFLP) analyses. Documentation of a combination of restriction fragment patterns which are characteristic of MCF-7 has been facilitated by an analysis of DNA from an early-passage of MCF-7 from the Michigan Cancer Foundation (MCF). In addition, such early passage cells and other MCF-7 lines also share a unique amplification of the N-ras oncogene. Using these criteria and karyotypic analysis it can be shown that a sample of MCF-7 obtained from the American Type Culture Collection (ATCC) was derived from a different individual than was the original MCF-7 cell line. It is important that researchers verify the relationship of current cell lines to the original MCF-7. Furthermore, the techniques described here provide a powerful tool which may be used to assess the identity of cell stocks.

A simplified method for passage and long-term growth of human mammary epithelial cells.

A method is described for culturing human mammary epithelial cells in primary culture and allowing more than 50 generations and a 1000-fold increase from starting inocula without need of enzymatic transfers. Organoids dissociated from breast tissue are plated in medium containing 1.05 mM Ca++ to effect attachment and growth to monolayer density. Medium is then switched to one containing 0.06 mM Ca++ to overcome "renewal inhibition" and to stimulate growth. In low Ca++ media, primary cultures become a long-term, continuous source of free-floating viable cells free of fibroblasts. A fundamental requirement for extended growth in primary culture is maintaining calcium levels at approximately 0.06 mM. Above 0.06 mM Ca++, cells divide only 3 to 4 times in primary cultures before terminal differentiation occurs. At 0.06 mM Ca++, cells continue to divide for periods of time determined partly by feeding schedule, but up to 6 mo. and 50 generations of (linear) growth. Cells released from monolayer were greater than 90% viable and yielded 10(5) cells/cm2 of attached cells every 72 h. Free-floating single cells readily replated and cloned, when transferred, without need of trypsin for dissociation. Long-term free-floating cells were typical mammary epithelium: they formed domes and exhibited renewal inhibition, they produced ductlike formations in collagen gels, they contained epithelium-specific keratin filaments, and they were diploid.

A common mouse mammary tumor virus integration site in chemically induced precancerous mammary hyperplasias.

Mammary carcinomas can be induced by chemical and hormonal as well as viral carcinogens. Irrespective of the class of inducer, these tumors develop in discrete stages, of which alveolar hyperplasia is one of the earliest identifiable. Since carcinogenesis by the mammary tumor virus is now thought to involve proviral activation of adjacent cell genes at specific loci, we sought to determine if a similar mechanism also played a role in chemical and hormonal carcinogenesis and if its role was stage specific. Three high-tumor-incidence BALB/c hyperplastic alveolar nodule outgrowths of two different etiologies were found to have exogenous mouse mammary tumor virus proviruses integrated at the same site in the genome. This common site of integration is not within the bounds of the int-1 and int-2 loci into which proviruses detected at these loci are clustered in MMTV-induced mammary tumors. All three HANs are commonly impaired in end-point differentiation. We propose that mouse mammary tumor virus integration at this site is responsible for a specific abnormality in differentiation associated with the preneoplastic phenotype.

Calcium regulation of normal human mammary epithelial cell growth in culture.

The concentration of Ca++ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca++ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of alpha-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca++ to less than 0.08 mM. The reduction of Ca++ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca++ on growth and differentiation were specific (Mg++ and Mn++ variations were without effect) and reversible and in many respects resembled Ca++ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca++-induced coupling and uncoupling of proliferation and the program of differentiation, we proposed that Ca++ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu.

Evidence for a direct growth-stimulating effect of estradiol on human MCF-7 cells in vivo.

The MCF-7 continuous line of human breast cancer cells requires that athymic nude mice receive supplemental estrogen so that inocula can produce progressively growing tumors. Although these cells contain a typical estrogen receptor complex, the lack of consistent growth stimulation induced by estrogens added to in vitro culture systems has raised the question as to whether this class of hormones acts directly upon the cells or induces a second message produced in other tissues. The present experiments were designed to test the effect of estradiol on the growth of these cells in vivo by exposing them directly to the hormone prior to its absorption into the hepatic portal circulation and subsequent metabolic inactivation. Tumor fragments that were placed next to an estradiol-containing pellet in the spleen grew to produce grossly evident tumor masses, whereas those in the subcutis of the same animals did not, although some minute residua did remain. In the splenic tumors, the mitotic index of the MCF-7 cells immediately adjacent to the estrogen pellets was 2.4 times that of cells on the other side of the same tumor and 3.5 times that of those in the minute s.c. residua. We interpret these data as indicating that in vivo estradiol is acting directly upon the MCF-7 cells to increase their rate of proliferation rather than to initiate the production of a second message to be released into the circulation. Additionally, it was found that s.c. tumors that were decreasing in volume subsequent to withdrawal of systemic estrogen still contained dividing neoplastic cells but with a lower frequency than that seen in progressively growting tumors stimulated with estradiol. This finding indicates that MCF-7 cells can proliferate in vivo in the absence of a substantial amount of estrogen but only at a rate insufficient to sustain progressive tumor growth.

Renewal inhibition of human mammary cell growth in vitro: cortisol and the recruitment of cells to terminal differentiation.

Cortisol and insulin stimulated exponential growth of normal human mammary epithelium in short-term monolayer culture. The response of cells depended on their organization into "growth units" on the surface of the culture dish; single cells did not respond. Growth of cells in the units ceased after only 3-4 doublings, ending in terminal differentiation. The 3-4 divisions that occurred in response to insulin and cortisol and that resulted in terminal differentiation, were not inhibited by short-range signals normally transmitted at population confluence. When growing above confluence density in response to hormones, cells reduced volume to accommodate the "terminal differentiation" divisions while still largely preserving a monolayer. The longevity of populations of normal cells (9-14 divisions), which occurred in 3 or 4 passages, exceeded the average longevity of individual cells in one passage (3-4 divisions). This disparity between real and apparent longevity was due to the inhibition of growth of divisional cells within growth units, which occurred in concert with terminal differentiation of other cells in these units. Inhibited cells could be recruited to undergo terminal differentiation divisions in response to cortisol and insulin, but only when the growth units were disrupted and terminally differentiated cells were eliminated, which occurred at subculture. We refer to the inhibition of growth that occurs in growth units as "renewal inhibition" to distinguish it from population-wide "confluence inhibition" and to emphasize three other aspects: (1) it occurred in terminally differentiating growth units; (2) it occurred in the continued presence of an inductive hormonal stimulus for differentiative growth; and (3) it conserved less differentiated cells for recruitment to terminal differentiation. There are parallels between renewal inhibition in vitro and the signals that restrain growth of mammary cells in 'growth buds" in vivo to preserve their capacity for multiple cycles of secretory differentiation. Differences in the behavior of normal and malignant breast cells in vitro suggest that renewal inhibition, rather than confluence inhibition, may be an important locus of growth control alteration in malignant transformation.