ALDH1A1_V01_JasonMorris_23Mar2015

ALDH Research Project and Experiments

Report 1 of 2

ABSTRACTThe source of our ALDH1A1 protein is from human breast adenocarcinoma cells from the Michigan Cancer Foundation and are known as MCF-7 cells. The upstream process of ALDH1A1 included: extracted mRNA to create cDNA via reverse transcriptase; sequenced to ensure presence of the ALDH1A1 gene and then run through PCR using taq-polymerase to create A-overhangs which were T-A cloned into vector pCR3.1 Uni using the RecA-; streaked the competent bacteria onto antibiotic-containing LB-agar plates; subcloned into expression vector pET Blue 1 and transformed into E. coli BL21DE3 and streaked on ampicillin LB-agar plates to be used for blue-white screening; as well as sequenced to ensure ALDH1A1 presence. A scale up procedure of the fully transformed culture using 300 ml LB-broth was initiated for protein translation in future experiments.

Jason Morris, Antony CraneThis project and report is towards fulfillment of requirements for the Biochemistry Laboratory Course during the Spring semester 2015 at Minneapolis Community and Technical College, under the advising of Dr. Rekha Ganaganur

23 March, 2015 ALDH Report One Page 1 of 12

Introduction:

ALDH stands for aldehyde dehydrogenase and belongs to a superfamily, composed of 18 familes

and 37 subfamilies, of NAD(P)+-dependent enzymes with similar primary structures, helping to oxidize

endogenous and exogenous aliphatic and aromatic aldehydes. The 1A1 naming system consists of an

Arabic number representing the family, a letter representing the subfamily and an Arabic number

representing the gene within the subfamily. The role of dehydrogenases, and substances that

dehydrogenate, is to oxidize molecules via removal of hydrogen ions. The functional groups that undergo

dehydrogenation in ALDH are aldehydes where they are converted to carboxylic acids. Nine families of

ALDH can be found in humans.

Subfamilies found in humans include ALDH1A, ALDH1B, ALDH2, ALDH3A, ALDH3B,

ALDH4A, ALDH5A, ALDH6A, ALDH7A, ALDH8A and ALDH9A. ALDH1 is found in neurons and

red blood cells, oxidizing retinaldehyde and aliphatic hydrides. ALDH2 is a mitochondrial enzyme and

handles acetaldehyde detoxification. ALDH3 helps to oxidize aromatic and fatty aldehydes and is found

in the human cornea. ADLH4 is also found in mitochondria, oxidizing y-semialdehydes while disrupting

degradation of proline. ALDH4 deficiency has been a cause of type II hyperprolinemia, mental

retardation and convulsion. ALDH5 oxidizes succinic semialdehydes. ALDH6 assists in degrading valine

and pyrimidine and also is involved with CoA. ALDH7 helps in alcohol metabolism and protects cells

from osmotic stress. ALDH8 reacts with 9-cis-retinal (Source: Wikipedia). ALDH9 breaks down

aminoaldehydes. ALDH10 – 18 are, respectively, found in plant, bacteria and yeast cells (source:

Vasiliou, Bairoch, Tipton and Nebert).

In general, ALDHs help break down aldehydes to carboxylic acids, where they are used in

cellular metabolisms. The presence of ALDH is essential to the breakdown of alcohol in the body, as it

works with alcohol dehydrogenase to convert alcohol to acetic acid and water. ALDH, while an essential

enzyme works against the interests of the body by promoting resistance in cancer cells treatable by a class

of drugs known as oxazaphosphorines (Source: Sloderbach, Górska, Sikorska, Misiura and Hladon).

The cell line we’re using for our ALDH project comes from human breast adenocarcinoma cells

from the Michigan Cancer Foundation. The cells, called MCF-7 cells, were taken from a cancer biopsy

from a patient via informed consent. The cancer drugs cyclophosphamide and ifosfamide, both

oxazaphosphorines, are both used to treat solid tumors and blood malignancies. However, ALDH

expression is one factor tied to resistance to the above-mentioned drugs. It is the hope of researchers,

through inhibition of the gene expression of ALDH via selective antisense oligonucleotides, that the drug

resistance can be thwarted (Source: Sloderbach, Górska, Sikorska, Misiura and Hladon). To clone the

ALDH1A1, we initially took the total mRNA from the MCF-7 cells and applied reverse transcriptase to


create cDNA. The ALDH1A1 gene was isolated from the cDNA and ran through PCR via taq-polymerase.

Later the ALDH1A1 gene was cloned using the T-A cloning method in vector pCR3.1 Uni, taken from the

pUC19 plasmid. Since the pUC19 plasmid has antibiotic resistance to several antibiotics, to test that the

pUC19 plasmid was transformed into competent recA- E. coli, the bacteria were streaked onto LB-agar

plates containing the same antibiotics. Colony-PCR was then applied to surviving colonies and two PCR

tests were performed. One involving the original set of primers and the other with a different set of

primers specific to the internal region of the ALDH1A1 gene. The gene was later confirmed by

sequencing.

A subcloning process is when an already existing cloned gene is then “subcloned” into yet

another vector.3 Although this mechanism isn’t always a necessity, some vectors are better utilized for a

particular purpose than others. For the sake of this experiment, the ALDH1A1 gene was subcloned from

cloning vector pCR3.1 Uni into the T-overhang containing expression vector pET Blue 1 with the aim of

high protein expression.4 This trait of increased protein expression is one reason why expression vectors

are commonly subcloned from previous cloning vectors. Another is because this plasmid vector is

modified for regulatory sequences that promote efficient transcription of gene of interest. Subcloning was

done with E. coli strain BL21DE3pLys using a fusion Histidine (His) tag. The His-tag facilitates

purification of expressed protein through recognition via chromatographic analysis. The new ligated DNA

E. coli were streaked on LB-agar plates with ampicillin and X-gal to be identified with blue/white

screening for successful ALDH1A1 clones. A sequencing was also completed to ensure the existence of

ALDH1A1.

Isopropyl-I-D-thiogalactoside (IPTG) was introduced to the culture of recombinant E. coli

BL21DE3pLys as an inducing agent for lacZ operon. lacZ operon is the promoter responsible for β-

galactosidase production (i.e. high protein yield). The amount of IPTG to induce with was calculated via

UV-Visible spectrophotometer prior to induction. Bacterial concentration was not ideal and underwent an

extended culture in order to achieve a higher density, making more protein for scale-up in the future. This

protein is not extracellular, making harvesting of supernatant undesirable. The induced bacterial pellet

was harvested in its stead. A cell lysis buffer tris, with amounts of protease inhibitors EDTA and PMSF

as well as membrane permeability inducer triton X-100 was used to cryogenically store the induced

bacteria at -80 °C. Since expression vector pET Blue 1 has a His-tag, immobilized metal ion

chromatography (IMAC) will ensure proper characterization and purification further downstream.

Through this scale up process a large amount of ALDH1A1 protein will be utilizable for future

experiments.


Materials and Methods:

Various technical procedures conducted with the following instruments:

-TEKNOV Agar; Lot # L911023H1201; Expires 27 August, 2017

- Difco LB Broth; Lot #: 2171196, Reference # 244620, Expires 31 May, 2017

- Fisher Scientific Plate; Model 2052FS; Serial # 1649080344320

- GETINGE Steam Autoclave; Serial # 08H08850; Checkup done 7 April, 2014

-GENMATE Micropipettor Set #6; Serial # 840180045

-VWR Shaking Incubator; Model 1585; Utilized 37 ° C at 200 rpm

- Bio-Rad Labs SmartSpec Plus Spectrophotometer; Serial # 273 BR 07396 ; Utilized 600 nm

- Beckman Coulter Allegra X-22 R Centrifuge; Serial # ALB03F019; ID # BIOT-0118; utilized for 30 minutes at 4000 rpm

-Eppendorf Centrifuge; Model 5415D; Serial # 5425-26984; ID # BIOT-0115

-Isotemp Fisher Scientific; Model 2052FS Dryblock heater; Serial # 1649080344320

-Cryogenic Storage Freezer; Utilized at -80 ° C

-Generic Eppendorf tubes

-Various sized class-A glassware

-Graduated pipettes and pipette aids

-Assorted glassware applicable to these experiments

-Inoculating loop; Utilized for inoculating and culture retrieval

-Laminar Flow Bench

-Level 2A Biological Safety Cabinet (BSC)


The procedures conducted on this project were carried out as follows:

All glassware, materials, and solutions unless thermally labile were sterilized via autoclave for

LB agar and broth preparations. January 22nd, thermally labile antibiotics ampicillin and chloramphenicol

were introduced to the media after autoclaving, and the media were stored at 4 °C for the following week.

The following week, on January 29th an LB-agar plate was streaked from the master stock of expression

vector pET-Blue transfected E. coli BL21DE3pLysS containing ALD1A1. Culture incubated no longer

than 18 hours to prevent antibiotic degradation and satellite colony formation. Small scale liquid culture

initiated February 5th by inoculating one cloned E. coli BL21DE3pLysS colony aseptically in a level-2A

BSC into 15 mL of ampicillin and chloramphenicol containing LB-broth(p.27 of notebook). At this point

inducing agent IPTG was not yet added because further mitotic division was desired for larger protein

yield on scale up process. The tube was incubated no more than 20 hours in a VWR shaking incubator at

37 °C and 200 rpm. The LB-broth stock and LB-agar plate was stored at 4 °C in a cold room. Scale up

process was initiated February 12th by first preparing an undiluted and 1:10 culture:LB-broth diluted

sample of culture from the small scale culture in polyacrylic cuvettes. The remainder of the small scale

culture was aseptically transferred into 300 mL of LB-broth and incubated in VWR shaking incubator at

37 °C at 200 rpm for approximately 2 hours to increase cell growth prior to IPTG induction. The

undiluted, diluted, and a blank LB-broth cuvette were spectrophotometrically analyzed at 600nm for

optical density (OD) to determine cell density. Optimum OD for protein production was 0.6. Once the OD

reading was between 0.3-0.6, inducing agent IPTG was added to the large scale 300 mL culture in a level-

2A BSC and incubated in VWR shaking incubator at 37 °C at 200 rpm for a maximum of 16 hours. On

the 19th of February the bacterial culture was spun down to pelletize it due to intracellular protein

production. After decanting off the supernatant, an EDTA, PMSF (protease inhibitors), and containing

lysis buffer was added and the culture was cryogenically stored at -80 °C. A small 100 μL sample of both

the uninduced and induced culture were stored at -20 °C.


Results:

The LB-media was successfully prepared. After culturing E. coli BL21DE3pLysS on a LB-agar

plate, the surviving colonies had the following morphologies and arrangements: white and creamy grey in

color, smooth texture, and raised off the plate. The survival of E. coli BL21DE3pLysS indicated that

ALDH1A1 containing pET Blue 1 expression vector was successfully transformed. A week after

inoculating the LB-broth with BL21DE3pLysS, growth in the broth indicated the transformation was

successful once more. After subtraction of the blank, the undiluted culture OD reading at 600nm was

2.299 with 1.15x109 cells/mL, and the diluted was 0.254 with 1.27x108 cell/mL. After accounting for the

dilution factor, the diluted culture was 2.54, indicating that the undiluted OD reading was reasonable.

Based on culture calculations, the amount of culture available was less than impeccable. The scale-up

culture was then initiated with an OD of 0.10730 at 600 nm. After 2 hours incubation, 0.730 OD was

reached indicating that cell density was high enough to induce the scale-up with IPTG, guaranteeing a

high protein yield. After storing the culture for 7 days in a cold room, it was retrieved and spun down to a

pellet. Refer to cover page picture for visual notations. This pellet indeed contained ALDH1A1 and was

then cryogenically stored in a PMSF lysis buffer at -80 °C.

Discussions and Conclusions:

Section I:

New concepts that were developed in the biotechnological biochemical laboratory in spring, 2015

were cloning processes, both upstream and downstream. The cloning vector pCR3.1 Uni and the pET

Blue 1 expression vector both have unique purposes that are not learned in the many science courses. This

laboratory is privileged to contain these experiments. Dr. Rekha’s direct support and guidance made

possible the hands on study of inducing agent IPTG and how it activates the promoting region of

laZoperon to initiate protein synthesis. Education of new concepts, vocabulary, and processes can be

conveyed in a classroom, but are best taught in a laboratory setting such as this. From the over expression

of ALDH1A1 in MCF-7 cells, to the derivation of those cells from human breast adenocarcinoma cells, to

both the upstream and downstream cloning processes and ultimately scaling up protein production, these

are invaluable skills and knowledge to have.

Section II: Paragraphs from scientific literature articles

The expression of different phenotypes from the same gene and the induction/inhibition of

enzymes such as ALDH were studied as they relate to disease and other biological effects.5 Sensitivity to

toxic chemicals varies between individuals and ethnicities, largely caused by differences in the

metabolism of different chemicals through the body. However, conflicting reports suggest a much more


limited correlation between genetic predisposition and environmental exposure. Further studies are

needed. This study one-ups the study by Hsu, et. al.

Clones of ALDH1 and ALDH2 were isolated from a human liver cDNA library and grown in

phage λgt11.6 Since the sequence of ALDH2 was unknown, they sequenced a portion of it and

discovered, in comparison to ALDH1, a 66% similarity in the cDNA and a 69% similarity in the proteins.

They also determined that there were racial differences between Asians and Caucasians in how they

metabolize alcohol, finding that while Caucasians have “usual” amounts of ALDH1 and ALDH2 in their

livers, 50% of Asians have only the ALDH1 enzyme and are missing the ALDH2 enzyme entirely.

ALDH1 genes impart resistance to high-dose chemotherapy in cancer cells via overexpression of

the ALDH1 gene.7 To test it, they gave cyclophosphamide-related drugs to human and murine cells. The

resistant phenotype confirmed an overexpression of ALDH1 genes. Used it to legitimize testing ALDH1

gene transfer to protect bone marrow cells. Since cyclophosphamide causes a decrease in peripheral blood

cell counts, the ultimate goal of creating a cyclophosphamide-resistant bone marrow allowing for

continued cyclophosphamide administration due to less blood loss.

The ALDH superfamily of enzymes plays an essential role in oxidizing aldehydes into less toxic

substances.8 Mutations in the ALDH gene lead to the absence, deficiency or deactivation of ALDH

proteins. From studies of ALDH genotypes, it’s apparent that an individual’s ALDH genotype should be

taken into account to design an effective treatment for diseases and other clinical treatments.

Studies were done showing that ALDH1A1 in the cornea and lens and ALDH3A1 in the cornea

protect mice against cataracts through detoxification of aldehydes and limiting free oxygen radicals that

would lead to protein cross-linking and clumping.9 In addition, the absence of ALDH3A1 in the lens led

to lens opacification, due to the ALDH3A1 absorbing UV light.

BRCA1 protein is responsible for the differentiation of mammary stem cells.10 If a BRCA1

mutation occurs, an enlarged stem cell component might also be present, and those stem cells could be the

origin of BRCA1 related breast cancer. In the study, the researchers used ALDH1 as a marker of both

mammary stem cells and breast cancer stem cells, and compared ALDH1 expression in malignant tissue

of BRCA1 mutation carriers and non-carriers. They found that BRCA1 related breast cancers showed

more ALDH1 expression, meaning that they also have a greater cancer stem cell component. As a result

of this study, ALDH1 may be used as both a marker and target of BRCA1 related breast cancer.

The research discusses 12 ALDH genes that have discovered in humans at the time of the paper.2

The genes are responsible for oxidation of aldehydes. It traces the evolutionary process from the first


occurrence of ALDH1/2/5/6 gene clusters 800 million years ago to the duplication in the ALDH3/10/7/8

gene 300 million years ago, with separations of ALDH3/10 and ALDH7/8 happening with the appearance

of mammals.

ALDH is considered to be a Phase II enzyme.11 As such, it metabolizes drugs into

pharmacologically active compounds as well as plays a role in the biotransformation of endogenous

compounds and xenobiotics to forms that are easier to dispose. While the reactions are usually towards

detoxification, the products formed may be produce ill effects. A number of procarcinogens get converted

by Phase II enzymes into intermediates that can act as chemical carcinogens and mutagens. As a result,

understanding Phase II biotransformation is essential to understanding human toxicology.

As it relates to ALDH, the article states that polymorphism of ALDH2 confers differing abilities

to process acetaldehydes, and thus contributes to predispositions toward an individual’s ability to process

alcohol.12 While the articles mentions gender and race differences both in the ability to process alcohol

and avoiding alcoholic liver diseases, it suggests that the differences are not necessarily related to a single

gene, but rather a combination of genes working together. Through further study, the researchers hope

that “high risk” individuals could be identified and given appropriate counseling.

Vitamin A deficiency can be a double-edged sword, in that retinoic acid (RA), acting as the most

active form of vitamin A, is also one of the most potent teratogens.13 As a result, in the developing

embryo, concentrations of both endogenous and exogenous RA are highly regulated, low in some areas

and high in others. However, most of the RA is oxidized from retinaldehydes by aldehyde

dehydrogenases. Ultimately, aldehyde dehyrdogenases play a protective role in eye development by

oxidizing free aldehydes. The resulting RA-mediated control may help in eye development in addition to

other processes.

The resistance of cancer cells to cyclophosphamide has been documented and corresponding

increased expression of ALDH1A1 have been linked to the diagnosis of other cancers throughout the

body.14 However, low expression of ALDH1A1 do not present a higher rate of survival in individuals

with pancreatic cancer. The researchers found that, while cyclophosphamide resistance is directly linked

to ALDH1A1 overexpression, other forms of chemotherapy work more effectively with higher levels of

ALDH1A1 expression in the cancer cells, meaning that overexpression of ALDH1A1 is essential for the

effectiveness of those drugs.

ALDH bright (ALDHbr) cell populations, consisting of stem cells with high expression of

ALDH, have been isolated from several different sources using the Aldefluor method.15 The ALDHbr

cells have important roles in hematopoietic transplantation and in ischemic heart disease. Preclinical trials


of transplantation of the cells to treat tissue repair are already underway and other uses may emerge from

the results of those trials. However, the complexities involved in preparation and culturing of the cells for

specific tissue repair treatments will be challenging. In addition, how the ALDHbr cells fare in

comparison to other treatments when exposed to cytokines is yet undetermined.

Other highlights:

ALDH is classified as a drug-metabolizing enzyme due to its tendency to detoxify aldehydes and

other toxins when they appear in the body, even if the aldehydes and other toxins are part of the

mechanism of drugs meant to kill cancer cells.16 Other drug-metabolizing enzymes include cytochrome

P450, cytochrome b5, and NADPH-cytochrome P450 reductase, all of which are considered Phase I and

typically activate carcinogenic and/or mutagenic agents. Phase II drug-metabolizing enzymes, including

glutathione S-transferase, aryl sulfatase, UDP-gluceronyl transferase inactivate carcinogens into less

harmful metabolites. Phase I enzymes introduce reactive or polar groups into chemicals foreign to the

body, creating acids, amines and alcohols, while Phase II enzymes take the same modified chemcials and

conjugate them into less toxic chemicals (7). As demonstrated by ALDH creating resistance to

cyclophosphamides, drug/chemical inactivation is not always a good thing. Other chemicals that ALDH

enzymes act on include: retinol, retinal, LPO-derived aldehydes and other aldehyde-type metabolites.

Other roles and implications of ALDH in the body include: development and maintenance of

epithelial tissues throughout the human body (ALDH1A1), RA synthesis in developing tissues

(ALDH1A2), embryonic development, eye development, olfactory bulb development, hair follicle

development, forebrain and cerebral cortex development (ALDH1A3), DNA replication and repair

(ALDH1L1), mitochondrial function (ALDH2), protecting the eye against UV stress, regulating cell

proliferation and the cell cycle (ALDH3A1), fatty aldehyde metabolism (ALDH3A2), protecting the

brain through detoxification of other aldehydes (ALDH3B1), DNA repair and cell survival (ALDH4A1),

valine and pyrimidine catabolism (ALDH6A1), lysine catabolism (ALDH7A1), and are found throughout

the body (Marchitti, et al). In stem cells, ALDH expression is especially high, helping with tissue repair in

organs throughout the body, including the heart.

An additional source for cloning the ALDH1 and ALDH5 is baker’s yeast (Saccharomyces

cerevisiae). For the purposes of one study, the yeast were first grown in yeast extract, peptone and

glucose medium then spread on SD plates. A genetic fragment containing the ALDH was them run

through PCR to amplify the gene, using the following primers: 5’ primer TTTGAACATATGGCT(C or

A)TTCACA(C)GGT(C)TCC(T or G)ACT, and 3′ primer, TTTGGATCCA(C)ACTGGA (C or

T)CCGAAA(G)ATT(C)TCT(C)TC. The 500 bp fragments were digested with Nde I and Bam H1 and

then cloned into pT7-7 vector.


Acknowledgements:

We would like to thank the biotechnology department staff overall for their invaluable

contributions to our project including Kelly Stedman, Dylan Stenvik, Shequaya Braodus, Greggory

Gilles, and Timothy Crushshon. A special thanks goes out to Dr. Rekha Ganaganur for her diligent

and committed leadership of the project and research we have conducted over the last few months!

THANK YOU!


Sources:

1. Heerma van Voss, Marise R. et al. “Expression of the Stem Cell Marker ALDH1 in BRCA1 Related Breast Cancer.” Cellular Oncology (Dordrecht) 34.1 (2011): 3–10. PMC. Web. 21 Mar. 2015.

2. Rzhetsky, Andrey et al. “Human aldehyde dehydrogenase gene family” Beckman Research Institute of the City of Hope, Duarte CA, USA (1997): 549-557. Print.

3. Dr. Rekha, Ganaganur. "ALDH Outline 1: Upstream Processes of the Inquiry-Based Guided Learning Project." Biotechnology Department, Biochemistry Laboratory Course 1 (2014): 1-5. Minneapolis Community and Technical College. Web.

4. Dr. Rekha, Ganaganur. "ALDH Outline 2: Subcloning Into Expression Vector: Upstream Process-2 of the Inquiry Based Guided Learning Project ALDH.” Biotechnology Department, Biochemistry Laboratory Course 1 (2014): 1-9. Minneapolis Community and Technical College. Web.

5. Nakajima, T. and Aoyama, T. “Polymorphism of Drug-Metabolizing Enzymes in Relation to Individual Susceptibility to Industrial Chemicals”. Industrial Health. 2000, 38. 143-152.

6. Hsu, L. C., Tani, K., Fujiyoshi, T., Kurachi, K., Yoshida, A. “Cloning of cDNAs for human aldehyde dehydrogenase 1 and 2”. Proc. Natl. Acad. Sci. USA. Vol. 82, pp. 3771-3775, June 1985.

7. Magni, M., Shammah, S., Schiro, R., Mellado, W., Dalla-Favera, R., and Gianni, A. M. “Induction of cyclophosphamide-resistance by aldehyde-dehydrogenase gene transfer”. Blood. 1996 87:1097-1103.

8. Marchitti, S.A., Brocker, C., Stagos, D. and Vasiliou, V. “Non-P450 aldehyde oxiding enzymes: the aldehyde dehydrogenase superfamily”. Expert Opin Drug Metab Toxicol. 2008 June ; 4(6): 697–720. doi:10.1517/17425250802102627.

9. Lassen, N., Bateman J.B., Estey, T., Kuszak, J.R., Nees, D.W., Piatigorsky, J., Duester, G., Day, B.J., Huang, J., Hines, L.M. and Vasiliou, V. “Multiple and Additive Functions of ALDH3A1 and ALDH1A1: CATARACT PHENOTYPE AND OCULAR OXIDATIVE DAMAGE IN Aldh3a1(−/−)/Aldh1a1(−/−) KNOCK-OUT MICE”. J Biol Chem. 2007 August 31; 282(35): 25668–25676.

10. van Voss, M.R.H., van der Groep, P., Bart, J., van der Wall, E. and van Diest, P.J. “Expression of the stem cell marker ALDH1 in BRCA1 related breast cancer”. Cellular Onc. (2011) 34:3-10.

11. Jancova, P., Anzenbacher, P. and Anzenbacherova, E. “Phase II Drug Metabolizing Enzymes”. Not sure when it’s written or for what publication.

12. Day, C.P., Bassendine, M.F. “Genetic predisposition to alcoholic liver disease”. Not sure when it’s written (early 90s?) or for what publication.

13. Dräger, U.C., Wagner, E. and McCaffery, P. “Aldehyde dehydrogenase in the Generation of Retinoic Acid in the Developing Vertebrate: A Central Role of the Eye”. The Journal of Nutrition. 1998. 463S-466S.

14. Kahlert, C., Bergmann, F., Beck, J., Welsch, T., Mogler, C., Herpel, E., Dutta, S., Niemietz, T., Koch, M. and Weitz, J. “Low expression of aldehyde dehydrogenase 1A1 is a prognostic marker for poor survival in pancreatic cancer”. Cancer. 2011, 11: 275.

15. Balber, A.E. “Concise Review: Aldehyde Dehydrogenase Bright Stem and Progenitor Cell Populations from Normal Tissues: Characteristics, Activities, and Emerging Uses in Regenerative Medicine”. Stem Cells. 2011;29;570-575.

16. Sreerama, L., Sladek, N. “Cellular levels of class 1 and class 3 aldehyde hydrogenases and certain other drug-metabolizing enzymes in human breast malignancies”. Clin Cancer Research. 1997 Nov;3(11):1901-1914.


17. Shewaita, S.A. “Drug-metabolizing enzymes: mechanisms and functions”. Curr Drug Metab. 2000 Sep;1(2):107-32.

18. https://courses.edx.org/c4x/DavidsonX/001x/asset/Ch_8_clip_4_summary.pdf 19. Wang, X., Mann, C.J., Bai, Y., Ni, L. and Weiner, H. “Molecular Cloning, Characterization, and

Potential Roles of Cytosolic and Mitochondrial Aldehyde Dehydrogenases in Ethanol Metabolism in Saccharomyces cerevisiae”. J. Bacteriol. February 1998 vol. 180 no. 4 822-830.

https://courses.edx.org/c4x/DavidsonX/001x/asset/Ch_8_clip_4_summary.pdf

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ALDH1A1_V01_JasonMorris_23Mar2015