Gertrude C. Buehring

CELL LINE CROSS-CONTAMINATION: HOW AWARE ARE MAMMALIAN CELL CULTURISTS OF THE PROBLEM AND HOW TO MONITOR IT?

SummaryHeLa was the first human cell line established (1952) and became one of the most frequently used lines because of its hardiness and rapid growth rate. During the next two decades, the development of other human cell lines mushroomed. One reason for this became apparent during the 1970s, when it was demonstrated that many of these cell lines had been overgrown and replaced by fast-growing HeLa cells inadvertently introduced into the original cultures. Although the discovery of these “HeLa contaminants” prompted immediate alarm, how aware are cell culturists today of the threat of cell line cross-contamination? To answer this question, we performed a literature search and conducted a survey of 483 mammalian cell culturists to determine how many were using HeLa contaminants without being aware of their true identity and how many were not using available means to ensure correct identity. Survey respondents included scientists, staff, and graduate students in 48 countries. HeLa cells were used by 32% and HeLa contaminants by 9% of survey respondents. Most were also using other cell lines; yet, only about a third of respondents were testing their lines for cell identity. Of all the cell lines used, 35% had been obtained from another laboratory instead of from a repository, thus increasing the risk of false identity. Over 220 publications were found in the PubMed database (1969–2004) in which HeLa contaminants were used as a model for the tissue type of the original cell line. Overall, the results of this study indicate a lack of vigilance in cell acquisition and identity testing. Some researchers are still using Hela contaminants without apparent awareness of their true identity. The consequences of cell line cross-contamination can be spurious scientific conclusions; its prevention can save time, resources, and scientific reputations.

Oral contraceptives and breast cancer. In vitro effect of contraceptive steroids on human mammary cell growth

The proliferative response of human mammary epithelial cells cultured in medium containing oral contraceptive steroids, singly and in combination, was measured. Cells came from 59 normal, nonmalignant atypical, and malignant breast tissue specimens. Growth of most cultures was stimulated by the estrogens 17β‐estradiol and ethinyl estradiol, and any combination containing ethinyl estradiol. Most (75%) malignant specimens were stimulated by one or more of the progestins, whereas none of the nonmalignant cells were. For two of the estrogen/progestin combinations found in many birth control pills, malignant cells were stimulated to grow faster than nonmalignant cells. It was concluded that oral contraceptive steroids can stimulate the growth of mammary epithelia in cell culture and might do the same in vivo. These data corroborate epidemiologic data which suggest oral contraceptives might act as tumor promoters, especially in the older user, who is more likely to have malignant cells already present in the breast. Cancer 59:281–287, 1987.

Screening for breast atypias using exfoliative cytology

A sample of 1744 self‐selected, mostly asymptomatic women was used to evaluate exfoliative cytology of nipple secretions as a screening technique for breast atypias. Results indicated: 1) overall frequency of breast fluid recovery was 49%, and related to age, phase of reproductive life, parity, history of lactation and birth control pill use at the time of sampling. 2) Frequency of satisfactory smears was 36% of breast fluid specimens, and related to subjects phase of reproductive life. 3) 2.8% of all satisfactory smears exhibited atypical cells. Biopsy of 4 subjects with atypical smears indicated 3 with nonmalignant epithelial atypias and 1 with early ductal carcinoma. Follow‐up of women who did not have atypical smears indicated 12/1092 (1.1%) with non‐malignant epithelial atypias and none with carcinoma. 4) The number of cases of malignant (1) and nonmalignant (7) breast disease detected was close to the expected number in the population sampled (1.98 and 7.25, respectively).

Effect of hormones on growth rates of malignant and nonmalignant human mammary epithelia in cell culture

SummaryThe individual effects of seven hormones on the in vitro growth rate of different classifications of human mammary epithelium were compared. Hormones used were: 17β-estradiol, estriol, progesterone, hydrocortisone, testosterone, prolactin, and growth hormone. Cell cultures included three established breast cell lines and primary monolayer cultures established form breast fluids and excised mammary tissue from 40 women and 4 men. Specimens comprised three classifications: normal, nonmalignant atypical, and malignant. Growth was quantitated in situ and expressed as population doubling time. Principal findings were: (a) estrogens, prolactin, and growth hormone stimulated growth of normal cells more frequently than growth of malignant cells, whereas testosterone and hydrocortisone stimulated growth of malignant cells more frequently than growth of normal cells; (b) cells cultured from nonmalignant atypias generally showed hormone response profiles intermediate between those of normal and malignant cells; (c) progesterone stimulated the growth of cells from malignant specimens but not the growth of cells from normal and nonmalignant atypical samples.

Human mammary cancer progression model recapitulates methylation events associated with breast premalignancy

IntroductionWe have previously identified a rare subpopulation of variant human mammary epithelial cells (vHMEC) with repressed p16INK4A that exist in disease-free women yet display premalignant properties, suggesting that they have engaged the process of malignant transformation. In order to gain insight into the molecular alterations required for vHMEC to progress to malignancy, and to characterize the epigenetic events associated with early progression, we examined the effect of oncogenic stress on the behavior of these cells.MethodsHMEC that express p16INK4A and vHMEC that do not, were transduced with constitutively active Ha-rasV12 and subsequently exposed to serum to determine whether signals from the cellular microenvironment could cooperate with ras to promote the malignant transformation of vHMEC. Epigenetic alterations were assessed using methylation-specific polymerase chain reaction (PCR).ResultsvHMEC expressing Ha-rasV12 (vHMEC-ras) bypassed the classic proliferative arrest that has been previously documented in normal fibroblasts following oncogenic stress, and that we also observe here in normal HMEC. Moreover, vHMEC-ras cells exhibited many additional alterations that are observed during progression to malignancy such as the generation of chromosomal abnormalities, upregulation of telomerase activity, immortalization following exposure to serum, and anchorage-independent growth, but they did not form tumors following orthotopic injection in vivo. Associated with their early progression to malignancy was an increase in the number of genes methylated, two of which (RASSF1A and SFRP1) were also methylated in other immortalized mammary cell lines as well as in breast cancer cells and tissues.ConclusionsWe have characterized a mammary progression model that recapitulates molecular and methylation alterations observed in many breast cancers. Our data suggest that concomitant methylation of RASSF1A and SFRP1 marks an early event in mammary transformation and may thus have prognostic potential.

Relative sensitivity and specificity of agar gel immunodiffusion, enzyme immunosorbent assay, and immunoblotting for detection of anti-bovine leukemia virus antibodies in cattle.

Three serologic methods for the detection of antibodies to bovine leukemia virus (BLV) were compared using the sera of 140 dairy cows. A widely used commercial agarose immunodiffusion screening assay and a commercial antibody capture enzyme immunosorbent assay were compared for sensitivity and specificity with immunoblotting as the standard. The immunoblot utilized the same antigen preparations that were provided in the commercial kits. The agarose immunodiffusion and the enzyme immunosorbent assay were comparable in the number of positive animals detected. However, the commercial screening kits failed to detect 39% (agarose immunodiffusion) and 35% (immunosorbent assay), respectively, of the animals determined serologically positive by immunoblot. These findings corroborate those of some other groups and emphasize the need for more sensitive tests to identify BLV positive cattle for culling or separation in order to create BLV-free herds.

Limited evidence of human papillomavirus on breast tissue using molecular in situ methods

Human papillomavirus (HPV) has been proposed as an etiologic agent of breast cancer based on numerous reports of high‐risk (oncogenic) HPV types in malignant breast tissues. However, most of those studies used standard and nested solution polymerase chain reaction (PCR) techniques, both of which are disadvantaged by vulnerability to laboratory contamination from positive control DNA and the inability to localize the signal to a specific cell type. To overcome these drawbacks, the authors of this report explored the use of in situ molecular methods of viral detection to reassess the frequency of HPV in malignant breast tissue.

Rare HRAS alleles and susceptibility to human breast cancer

The suggestion that inherited rare alleles at the HRAS oncogene locus might be associated with susceptibility to breast cancer led us to test linkage of HRAS and the neighboring region of 11p15 to breast cancer susceptibility in 12 high-risk families. Linkage could be excluded within 17 cM of HRAS; the lod score for close linkage to HRAS was -19.9. In addition, rare HRAS alleles segregated independently of breast cancer in 8 families in which both occurred. Among unrelated breast cancer patients not selected for family history, rare HRAS alleles were slightly, but not significantly, more frequent than among controls (0.11 vs 0.04, P = 0.11). The HRAS region of 11p is not the site of a primary alteration leading to breast cancer.

Inhibition of the estradiol-induced growth of cultured human breast cancer cells by the anti-estrogens tamoxifen, desmethyl-tamoxifen, 4-hydroxy-tamoxifen and enclomiphene

The growth effects of tamoxifen (T), desmethyl-tamoxifen (dMeT), 4-hydroxy-tamoxifen (OHT) and enclomiphene (Clo) on cultured human breast cancer cell lines have been related to published binding affinities for the estrogen receptor. Only in cells which were stimulated by estrogens did these anti-estrogens markedly inhibit growth. In both estrogen sensitive cell lines tested, 734 B and ZR 75.1, the anti-estrogen activity showed the identical rank: OHT much greater than Clo approximately equal to T = dMeT; this anti-proliferative potency agrees with reported affinities of these compounds for the estrogen receptor. In culture media containing defined amounts of estradiol we observed that a 10,000-fold molar excess of OHT was required to inhibit the estradiol-induced growth, but the estradiol-independent proliferation was not affected.

Mother to child transmission of HIV‐1 in a Thai population: Role of virus characteristics and maternal humoral immune response

The objective of this study was to investigate factors influencing mother to child transmission of HIV‐1 in Thailand, where HIV‐1 CRF01_AE, the major subtype in Southeast Asia, predominates. Samples from 84 HIV‐1 infected, anti‐retroviral treatment‐naïve, non‐breast feeding mothers, 28 who transmitted HIV‐1 to their babies (transmitters) and 56 who did not (non‐transmitters), were studied for maternal humoral immune response and virus characteristics. Maternal humoral immune response was measured by lymphocyte phenotyping; neutralizing antibodies to laboratory HIV‐1 MN strain and two clinical isolates; peptide binding antibody to gp41 and V3 from strains CRF01_AE, B, and MN; autologous antibodies; and quasispecies diversity. Virus characteristics studied were viral load, co‐receptor usage, and viral replication capacity. No significant difference between transmitters and non‐transmitters was found for any parameter of maternal humoral immune response. However, viral load and viral replication capacity were significantly higher in transmitters versus non‐transmitters and were not correlated with each other. This suggests that viral replication capacity may be a transmission factor independent of viral load, which is already well established as a risk factor for transmission of HIV‐1. All except four viral isolates used the CCR5 co‐receptor. This is one of few studies of vertical transmission in a population where HIV‐1 CRF01_AE predominates. The data suggest that in this population the maternal humoral immune response was not important in preventing transmission at parturition, but that virus characteristics were key factors, and that viral replication capacity may contribute to birth‐associated mother to child transmission of HIV‐1. J. Med. Virol. 81:768–778, 2009.